Analyte assay using particulate labels

ABSTRACT

Method for specific detection of one or more analytes in a sample. The method includes specifically associating any one or more analytes in the sample with a scattered-light detectable particle, illuminating any particle associated with the analytes with light under conditions which produce scattered light from the particle and in which light scattered from one or more particles can be detected by a human eye with less than 500 times magnification and without electronic amplification. The method also includes detecting the light scattered by any such particles under those conditions as a measure of the presence of the analytes.

RELATED APPLICATION

This application claims priority to Yguerabide et al., U.S. ProvisionalApplication No. 60/016,383, entitled “Analyte Assay Using ParticulateLabels”, filed Apr. 25^(th), 1996, which is hereby incorporated byreference in its entirety, including drawings.

BACKGROUND OF THE INVENTION

The following is an outline of relevant existing detection methods. Itis also a summary of relevant science to aid the reader in understandingthe details of the claimed invention. It should not be taken as anadmission that any of the cited art is prior art to the claims. Thecited art is hereby incorporated herein by reference so that the generalprocedures and methods in that art that are of use to practice of thepresent invention need not be rewritten herein. In particular, applicantincorporates those sections related to general methods of “binding-pair”methodology, and methods for measurement of light scattering herein.

Sensitive Analyte Assays

Binding-pair (also known as ligand-receptor, molecular recognitionbinding and the like) techniques play an important role in manyapplications of biomedical analysis and are gaining importance in thefields of environmental science, veterinary medicine, pharmaceuticalresearch, food and water quality control and the like. For the detectionof analytes at low concentrations (less than about 1 picomoleanalyte/sample volume analyzed) the use of fluorescent, luminescent,chemiluminescent, or electrochemiluminescent labels and detectionmethods are often used.

For the detection of low concentrations of analytes in the field ofdiagnostics, the methods of chemiluminescence andelectrochemiluminescence are gaining wide-spread use. These methods ofchemiluminescence and electro-chemiluminescence provides a means todetect low concentrations of analytes by amplifying the number ofluminescent molecules or photon generating events many-fold, theresulting “signal amplification” then allowing for detection of lowconcentration analytes.

In addition, the method of Polymerase Chain Reaction (PCR) and otherrelated techniques have gained wide use for amplifying the number ofnucleic acid analytes in the sample. By the addition of appropriateenzymes, reagents, and temperature cycling methods, the number ofnucleic acid analyte molecules are amplified such that the analyte canbe detected by most known detection means. The high level of commercialactivity in the development of new signal generation and detectionsystems, and the development of new types of test kits and instrumentsutilizing signal and analyte molecule amplification attests to theimportance and need for sensitive detection methods.

However, the above mentioned methods of signal and analyte moleculeamplification have associated limitations which makes the detection ofanalytes by these methods complicated, not easy to use, time consuming,and costly. Problems of interference of chemical or enzymatic reactions,contamination, complicated and multi-step procedures, limitedadaptability to single step “homogeneous” (non-separation) formats, andthe requirement of costly and sophisticated instrumentation are areasthat those in the art are constantly trying to improve.

Thus, there is a tremendous need for easy to use, quantitative,multi-analyte, and inexpensive procedures and instruments for thedetection of analytes. Such procedures, test kits, and instruments wouldovercome the disadvantages and limitations of the current methods ofsignal and analyte molecule amplification, and would be useful inresearch, individual point of care situations (doctor's office,emergency room, out in the field, etc.), and in high throughput testingapplications.

It is the object of the present invention to provide a new means to moreeasily detect one or more analytes in a sample to low concentrationsthan was previously possible. The present invention can detect lowconcentrations of analytes without the need for signal or analytemolecule amplification.

The present invention provides a signal and detection system for thedetection of analytes where the procedures can be simplified and theamount and types of steps and reagents reduced. The present inventionprovides for the quantitative detection of single or multiple analytesin a sample. The present invention also provides for substantialreductions in the number of different tests and amounts of samplematerial that are analyzed. Such reduction in the number of individualtests leads to reduced cost and waste production, especiallymedically-related waste that must be disposed of.

Light Scattering Detection Methods and Properties of Light ScatteringParticles

There is a large body of information concerning the phenomenon of lightscattering by particles, the use of particulate labels in diagnosticassays, and the use of light scattering methods in diagnostic assayswhich are now presented in the following discussion of relevant art noneof which is admitted to be prior art to the pending claims. This art isprovided as a background for understanding of the novelty and utility ofthe claimed invention.

The general study of light scattering comprises a very large field. Thephenomena of light scattering has been studied intensely for about thelast one hundred or so years and the applications of the knowledge oflight scattering to different aspects of human endeavor are wide andvaried.

The classical theory of light scattering by small, homogeneous, nonlight absorbing, spherical particles of a size of about {fraction(1/20)} or less the wavelength of the incident radiation was initiallydeveloped by Rayleigh. Later a more general phenomenological theory oflight scattering by homogeneous, spherical particles of any size andcomposition was developed by Mie. The Mie theory applies both to lightabsorbing and nonabsorbing particles. It has also been shown from Mietheory that the expressions of Rayleigh can easily be generalized so asto apply to particles which absorb light as long as the particles aremuch smaller than the wavelength of incident light. For these smalldiameter particles, Mie theory and the generalized Rayleigh theory givesimilar results. Light scattering (elastic) can be viewed from aclassical or quantum mechanical point of view. An excellent quantitativedescription can be obtained through the classical point of view.

A historical background as well as a description of the basic theoriesof scattered light and other electromagnetic radiation is provided inthe following references; Absorption and Scattering of Light By SmallParticles (1983), C. F. Bohren, D. R. Huffman, John Wiley and Sons; TheScattering of Light and Other Electromagnetic Radiation (1969), M.Kerker, Academic Press.

Further background information of the phenomenon of light scattering canbe found in the following publications.

Zsigmondy, Colloids and the Ultramicroscope—A Manual of ColloidChemistry and Ultramicroscopy,1914, John Wiley & Sons, Inc. is describedvarious light scattering properties of gold particles and other types ofparticles.

Hunter, Foundation of Colloid Science, Vol, I, 105, 1991, describes useof optical microscopes, ultramicroscopes, and electron microscopes inobservation of particles.

Shaw et al., Introduction to Colloid and Surface Chemistry, 2nd ed., 41,1970, describe optical properties of colloids and the use of electronmicroscopy, and dark field microscopy e.g., the ultramicroscope.

Stolz, SpringerTracts, Vol. 130, describes time resolve light scatteringmethodologies.

Klein and Metz, 5 Photographic Science and Engineering 5-11, 1961,describes the color of colloidal silver particles in gelatin.

Eversole and Broida, 15 Physical Review 1644-1654, 1977, describes thesize and shape effects on light scattering from various metal particlessuch as silver, gold, and copper.

Kreibig and Zacharias, 231 Z. Physik 128-143, 1970, describe surfaceplasma resonances in small spherical silver and gold particles.

Bloemer et al., 37 Physical Review 8015-8021, 1988, describes theoptical properties of submicrometer-sized silver needles and the use ofsuch needles is described in Bloemer, U.S. Pat. No. 5,151,956, where asurface plasmon resonance of small particles of metal to polarize lightpropagating in a wave guide is described.

Wiegel., 136 Zeitschrift fur Physik, Bd., 642-653, 1954, describes thecolor of colloidal silver and the use of electron microscopy.

Use of Particles, Light Scattering and Other Methods for Detection ofAnalytes

For about the last thirty-five years, metal particles including gold andsilver have been used as both contrast enhancement agents or lightabsorption labels in many different types of analytic and/or diagnosticapplications. The great majority of these applications fall under thecategory of cytoimmunochemistry studies which have used gold or silverenhanced gold particles as markers to study structural aspects ofcellular, subcellular, or tissue organization. In these studies, metalparticles are usually detected and localized by electron microscopy,including scanning, transmission, and BEI (backscattered electronimaging). These methods take advantage of the electron dense nature ofmetals or the high atomic number of metals to facilitate the detectionof the gold particles by virtue of the large numbers of secondary andbackscattered electrons generated by the dense metal (see; Hayat,Immunogold-silver staining reference Page 1 and Chapters 1, 6, 15; andHayat, Colloid Gold reference Chapters 1, 5, 7 and others).

There have been a few reports of the use of gold and silver enhancedgold particles in light microscopic studies. For example, in 1978 goldparticles were used as an immunogold stain with detection by lightmicroscopy. A review of the use of gold particles in light microscopy(See, Hayat, Immunogold-Silver Staining Reference Page 3) published in1995 discusses this 1978 work and presents the following analysis:

“Geoghehan et al. (1978) were the first to use the red or pink color ofcolloidal gold sols for light microscopical immunogold staining usingparaffin sections. In semithin resin sections red color of lightscattered from gold particles as small as 14 nm was seen in cellorganelles containing high concentrations of labeled antigens in thelight microscope (Lucocq and Roth, 1984). Since the sensitivity ofimmunogold staining in light microscopy is inferior in comparison withother immunocytochemical techniques, the former did not gain generalacceptance; the pinkish color of the gold deposit is difficult tovisualize.”

This paragraph is an indication of the state of understanding of thelight scattering properties of gold and other metal particles fordiagnostic and analytic studies. The paragraph specifically states “Insemithin resin sections red color of light scatter from gold particlesas small as 14 nm was seen in organelles containing high concentrationsof labeled antigens in the light microscope.”

However, with white light illumination, the scattered light from 14 nmgold particles is predominantly green. S1nce the particles appear red inthe light microscope this indicates that some interactions other thanpure light scattering are being detected. It is probable that the redcolor observed in the light microscope is predominantly transmittedlight and not scattered light. When the gold particles accumulatesufficiently at the target site in cells, tissue sections or some othersurface the red color due to transmitted light will be seen (see also;J. Roth (1983) Immunocytochemistry 2 p217; and Dewaele et al (1983) inTechniques in Immunochemistry Vol 2 p1, Eds. Bullock and Petrusz,Academic Press).

As mentioned in the above quote, it appears that the sensitivity ofimmunogold staining in light microscopy was believed to be inferior tothat of other methods, and the use of gold particles as markers forlight microscope detection did not gain general acceptance. In the 1995review book in Chapter 12, p198 by Gao and Gao is the following quote onthe same subject.

“Colloidal gold was initially used only as a marker for electronmicroscopy (EM), because of its electron dense nature and secondaryelectron emission feature (Horisberger, 1979). Direct visualization ofcolloidal gold in light microscopy (LM) was limited. The size ofcolloidal gold is too small to be detected at the light microscopelevel, although using highly concentrated immunogold cells may bestained red by this reagent (Geoghegan et al., 1978; Roth, 1982; Holgateet al., 1983.”

As mentioned in both of the above, the sensitivity of detection ofcolloidal gold with light microscopy was believed to be low. The methodof silver enhancement of gold particles was developed to overcome thisperceived drawback. The following is another quote from the 1995 reviewbook.

“The real breakthrough for immunogold staining for light microscopy camewith the introduction of silver-enhancement of colloidal gold particles(20nm) bound to immunoglobin in paraffin sections 5 microns (Holgate etal., 1983). This approach significantly enhanced the sensitivity,efficiency, and accuracy of antigen detectability in the lightmicroscope. Using IGSS, gold particles as small as 1 nm in diameter canbe visualized in the light microscope. Thin section subjected to IGSScan also be viewed with the light microscope, especially by using phasecontrast or epi-polarization illumination (Stierhof et al., 1992).”

The method of silver enhancement of gold particles is widely used. Theenhancement method transforms the marker gold particle into a largermetal particle or an even larger structure which is microns or greaterin dimensions. These structures are composed primarily of silver, andsuch enlarged particles can be more readily detected visually in thebright field optical microscope. Individual enlarged particles have beenvisualized by high resolution laser confocal and epipolarization lightmicroscopy. Id at 26 and 203.

However, even with the use of silver enhancement techniques, those inthe art indicate that this will not achieve the sensitivity andspecificity of other methods. For example, in the publication of Vener,T. I. et. al., Analytical Biochemistry 198, p308-311 (1991) the authorsdiscuss a new method of sensitive analyte detection called LatexHybridization Assay (LHA). In the method they use large polymerparticles of 1.8 microns in diameter that are filled with many highlyfluorescent dye molecules as the analyte tracer, detecting the boundanalytes by the fluorescent signal. The following excerpt is from thispublication:

“To assess the merits of LHA we have compared our technique with twoother indirect nonradioactive techniques described in the literature.The most appropriate technique for comparison is the streptavidincolloid gold method with silver enhancement of a hybridization signal,since this is a competing corpuscular technique. However, this method isnot very sensitive even with the additional step of silver enhancement:8 pg of λ-phage DNA is detected by this method as compared to 0.6 pg or2×10⁴ molecules of λ DNA detected by LHA on the nylon membrane.”

Stimpson et al., 92 Proc. Natl. Acad. Sci, USA 6379-6383, July 1995, areal time detection method for detection of DNA hybridization isdescribed. The authors describe use of a particulate label on a targetDNA which acts as a

“light-scattering source when illuminated by the evanescent wave of thewave guide and only the label bound to the surface generates a signal. .. . The evanescent wave created by the wave guide is used to scatterlight from a particulate label adsorbed at multiple DNA capture zonesplaced on the wave guide surface. S1nce an evanescent wave only extendsa few hundred nanometers from the wave guide surface, theunbound/dissociated label does not scatter light and a wash step is notrequired. The signal intensity is sufficient to allow measurement of thesurface binding and desorption of the light-scattering label can bestudied in real time; i.e., detection is not rate limiting. Thehybridization pattern on the chip can be evaluated visually or acquiredfor quantitative analysis by using a standard CCD camera with an 8-bitvideo frame grabber in {fraction (1/30)} of a second.”

Experiments were performed with 70 nanometer diameter gold particles and200 nanometer diameter selenium particles. More intense signals wereobserved with the selenium particles. The authors indicate

“A wave guide signal sufficient for single-base discrimination has beengenerated between 4 and 40 nm DNA and is, therefore, comparable to afluorescence signal system.”

This method uses waveguides and evanescent type illumination. Inaddition, the method is about as sensitive as current fluorescence-baseddetection systems. Particles of 70 nm diameter and larger are said to bepreferred.

Schutt et al., U.S. Pat. No. 5,017,009, describes an immunoassay systemfor detection of ligands or ligand binding partners in a heterogenousformat. The system is based upon detection of

“back scattered light from an evanescent wave disturbed by the presenceof a colloidal gold label brought to the interface by an immunologicalreaction. . . . Placement of the detector at a back angle above thecritical angle insures a superior signal-to-noise ratio.”

The authors explain that the immunoassay system described utilizesscattered total internal reflectance, i.e., propagation of evanescentwaves. They indicate that the presence of colloidal gold disruptspropagation of the evanescent wave resulting in scattered light whichmay be detected by a photomultiplier or other light sensor to provide aresponsive signal. They indicate that an important aspect of theirinvention is the physical location of the detector.

“The detector is ideally placed at an angle greater than the criticalangle and in a location whereby only light scattered backward toward thelight source is detected. This location thereby ideally avoids thedetection of superior scattered light within the bulk liquid medium.”

Total internal reflection of the incident beam is used to create theevanescent wave mode of illumination and the detection is performed onan optically-transmissive surface. The use of specialized apparatus ispreferred.

Leuvering, U.S. Pat. No. 4,313,734, describes a method for detection ofspecific binding proteins by use of a labeled component obtained bycoupling particles “of an aqueous dispersion of a metal, metal compound,or polymer nuclei coated with a metal or metal compound having adiameter of at least 5 nm.” The process is said to be especially suitedfor estimation of immunochemical components such as haptens, antigensand antibodies. The metal particles are said to have already been usedas contrast-enhancing labels in electron microscopy but their use inimmunoassays had apparently

“not previously been reported and has surprisingly proved to bepossible.

* * *

The metal sol particle, immunochemical technique, according to theinstant invention which has been developed can be not only moresensitive than the known radio- and enzyme-immunotechniques, but rendersit furthermore possible to demonstrate and to determine more than oneimmunological component in the same test medium simultaneously byutilizing sol particles of different chemical compositions as labels.”

Examples of metals include platinum, gold, silver, and copper or theirsalts.

“The measurement of the physical properties and/or the concentration ofthe metal and/or the formed metal containing agglomerate in a certainphase of the reaction mixture may take place using numerous techniques,which are in themselves known. As examples of these techniques there maybe cited the calorimetric determination, in which use is made of theintense colour of some dispersions which furthermore change colour withphysicochemical changes; the visual method, which is often alreadyapplicable to qualitative determinations in view of the above-noted factthat metal sols are coloured; the use of flame emissionspectrophotometry or another plasma-emission spectrophotometric methodwhich renders simultaneous determination possible, and the highlysensitive method of flame-less atomic absorption spectrophotometry.”

Two or more analytes in a sample are preferably detected by using flameemission spectrophotometry or another plasma-emission spectrophotometricmethod. The preferred method of detection for greatest sensitivity is byflame-less atomic absorption spectrophotometry.

Swope et al., U.S. Pat. No. 5,350,697 describes apparatus to measurescattered light by having the light source located to direct light atless than the critical angle toward the sample. The detector is locatedto detect scattered light outside the envelope of the critical angle.

Craig et al., U.S. Pat. No. 4,480,042 describes use of high refractiveindex particle reagents in light scattering immunoassays. The preferredparticles are composed of polymer materials. The concentration ofcompounds of biological interest was determined by measuring the changein turbidity caused by particle agglutination or inhibition ofagglutination. The preferred particles are of a diameter less thanapproximately 0.1 μ and greater than 0.03 μ. “Shorter wavelengths, suchas 340 nm, give larger signal differences than longer wavelengths, suchas 400 nm.”

Cohen et al., U.S. Pat. No. 4,851,329 and Hansen, U.S. Pat. No.5,286,452, describe methods for detection of agglutinated particles byoptical pulse particle size analysis or by use of an optical flowparticle analyzer. These systems are said to be useful for determinationof antigen or antibody concentrations. These methods use sophisticatedapparatus and specialized signal processing means. Preferred particlediameters are of about 0.1 to 1 micron in diameter for the method ofCohen and about 0.5 to about 7.0 microns in diameter for the method ofHansen.

Okano et al., 202 Analytical Biochemistry 120, 1992, describes aheterogenous sandwich immunoassay utilizing microparticles which can becounted with an inverted optical microscope. The microparticles were ofapproximately 0.76 microns in diameter, and were carboxylatedmicroparticles made from acrylate.

Other particle detection methods are described by Block, U.S. Pat. No.3,975,084, Kuroda, U.S. Pat. No. 5,274,431, Ford, Jr., U.S. Pat. No.5,305,073, Furuya, U.S. Pat. No. 5,257,087, and by Taniguchi et al.,U.S. Pat. No. 5,311,275.

Geoghegan et al., 7 Immunological Communications 1-12, 1978, describesuse of colloidal gold to label rabbit anti-goat IgG for indirectdetection of other antibodies. A light and electron microscope were usedto detect labeled particles. The gold particles had an average size of18-20 nanometers and bright field light microscopy was used. Forelectron microscopy, Araldite silver-gold thin sections were used.“Similar percentages of surface labeled cells were noted byimmunofluorescence and the colloidal gold bright field method.” 1-5particles per cell could be detected by electron microscopy but theauthors state that:

“Such small quantities of label were not detected by fluorescence or bybrightfield microscopy and may represent either non-specific and Fcreceptor bound GAD and GAM, where a low level of surface immunoglobulin(S.Ig) on the GAD and GAM treated cells.”

Hari et al., U.S. Pat. No. 5,079,172, describes use of gold particles inantibody reactions and detection of those particles using an electronmicroscope. 15 nanometer gold particles were exemplified. In thepreferred method, electron microscopy is used.

DeMey et al., U.S. Pat. No. 4,420,558, describes the use of a brightfield light microscopic method for enumerating cells labeled withgold-labeled antibodies. The method uses a light microscope in thebright field arrangement and magnifications of 500 or greater withimmersion oil lenses are used to count gold-labeled peroxidase negativecells. The visualization of the labeled-surfaces is based on theaggregate properties of the gold particles, which, under the indicatedcircumstances, undergo extensive patching, these patches on the cellsurface being resolvable with the method described. 40 nanometer goldwas found to give optimal results.

De Mey et al., U.S. Pat. No. 4,446,238, describes a similar bright fieldlight microscopic immunocytochemical method for localization ofcolloidal gold labeled immunoglobulins as a red colored marker inhistological sections. The method of Immuno Gold Staining (IGS) asdescribed by the authors

“In both procedures the end-product is an accumulation of large numbersof gold granules over antigen-containing areas, thus yielding thetypical reddish colour of colloidal gold sols.”

DeBrabander et al., U.S. Pat. No. 4,752,567 describes a method fordetecting individual metal particles of a diameter smaller than 200 nmby use of bright field or epi-polarization microscopy and contrastenhancement with a video camera is described. The inventors state:

“Typically, in the above mentioned procedures, the employed metalparticles have a diameter of from about 10 to about 100 nm. This is wellbelow the resolution limit of bright field microscopy, which isgenerally accepted to lie around 200 nm. It is therefore quite logicalthat all previously known visual light microscopic methods are limitedin their applications to the detection of immobilized aggregates ofmetal particles. Individual particles could be observed withultramicroscopic techniques only, in particular with electronmicroscopy.

It has now quite surprisingly been found that individual metal particlesof a diameter smaller than 200 nm can be made clearly visible by meansof bright field light microscopy or epi-polarization microscopy in thevisible spectrum, provided that the resulting image is subjected toelectronic contrast enhancement.”

In subsequent sections the authors state:

“Compared with existing diagnostic methods based on sol particle immunoassays, the present method has a much greater sensitivity. Indeed,existing methods are in general based on light absorption or scatteringby the bulk of absorbed or suspended metal particles. Obviously, theobservation of colour, e.g. on a blotting medium, requires the presenceof massive numbers of particles. In contrast therewith, the presentmethod makes it possible to observe and count single particles. Hence,the present method will largely facilitate the development of diagnosticblots for applications where existing, e.g. visual or calorimetric,techniques are too less sensitive, e.g. for the detection of Hepatitis.”

Schafer et al., 352 Nature 444-448, 1991, describes use of nanometersize particles of gold which could be observed using video enhanceddifferential interference contrast microscopy. A 40 nanometer diametergold particle was used.

DeBrabander et al., 6 Cell Motility and the Cytoskeleton 105-113, 1986,(and U.S. Pat. No. 4,752,567) describe use of submicroscopic goldparticles and bright field video contrast enhancement. Specifically, thecells were observed by bright field video enhanced contrast microscopywith gold particles of 5-40 nanometers diameters. They also state that

“individual gold particles, having a size smaller than plus or minus 100nanometers, adsorbed under glass or cells or microinjected in cells arenot visible in the light microscope. They are, however, easilyvisualized when using the capacity of a video camera to enhance contrastelectronically.”

The authors describe use of epi-illumination with polarized light andcollection of reflected light or by use of a “easier and apparently moresensitive way” with a transmitted bright field illumination usingmonochromatic light and a simple camera. The authors indicate that thegold particles can be easily detected with phase contrast microscopy.

“Unlike that which is possible with larger gold (usually 20-40 nm), evendense accumulations of 5-nm gold, e.g., on structures such asmicrotubules, are not visible in the light microscope. They do notproduce a detectable red colour. Recently, this has been corrected by aphysical development with silver salts, which increases the size of theparticles to produce an easily visible black stain.

* * *

We have described a method for localizing ligands almost at themolecular level. The method is new because it enables one for the firsttime to do this in the light microscope with discrete individual markersthat are unambiguously discernible from background structures. Becauseit is applicable even in living cells, one can thus follow the dynamicbehaviour of individual proteins. The method is because it combines twowell developed techniques: gold labelling and video microscopy. Most ofthe applications can be done with inexpensive video equipment the priceof which is less than most good 100 X oil objectives. Still, many morepossibilities arise when combining this with modern digital imagemanipulations. Some additional advantages are worth noting. Because thelabel consists of individual discrete markers, both manual and automatic(computer assisted) counting is easy and reliable. The small size of themarker minimizes problems of penetration and diffusion. The possibilityof changing the charge of the marker almost at will is helpful indiminishing nonspecific binding in any particular application.”

This method was termed by the authors “nanoparticle videoultramicroscopy or short nanovid ultramicroscopy.” A similar technologyis described in “Geerts et al., 351 Nature, 765-766, 1991.

The preceding discussions of the state of the art of light scatteringmethods, and the use of light scattering particles and methods in thefield of diagnostics clearly shows the limits of current methods ofanalyte detection and the novelty and great utility of the presentinvention. It is the purpose of this invention not only to overcome thepresent day limitations and disadvantages of light scattering-baseddiagnostic assays, but to also overcome the limitations anddisadvantages of other non-light scattering methods such as signal andanalyte molecule amplification. This invention as described herein iseasier to use, has greater detection sensitivity, and is capable ofmeasuring analytes across wider analyte concentration ranges than waspreviously possible. The present invention is broadly applicable to mostsample types and assay formats as a signal generation and detectionsystem for analyte detection.

SUMMARY OF THE INVENTION

The present invention features a new method for the detection andmeasurement of one or more analytes in a sample. The method is based onthe use of certain particles of specific composition, size, and shapeand the detection and/or measurement of one or more of the particle'slight scattering properties. The detection and/or measurement of thelight-scattering properties of the particle is correlated to thepresence, and/or amount, or absence of one or more analytes in a sample.The present invention is versatile and has utility in one form oranother to detect and measure one or more analytes in a sample.

The invention features a method for detection of one or more analytes ina sample by binding those analytes to at least one detectable lightscattering particle with a size smaller than the wavelength of theillumination light. This particle is illuminated with a light beam underconditions where the light scattered from the beam by the particle canbe detected by the human eye with less than 500 times magnification. Thelight that is scattered from the particle is then detected under thoseconditions as a measure of the presence of those one or more analytes.Applicant has surprisingly determined, by simply ensuring appropriateillumination and ensuring maximal detection of specific scattered light,that an extremely sensitive method of detection can result. The methodof light illumination and detection is named “DLASLPD” (direct lightangled for scattered light only from particle detected) by applicant.

The method and associated apparatus are designed to maximize detectionof only scattered light from the particles and thus is many times moresensitive than use of fluorophores, or the use of such particles inmethods described above. Such particles can be detected by using a lowmagnification microscope (magnifying at 2 to 500 times, e.g. 10 to 100times) without the need for any electronic amplification of the signal.In addition, methods are provided in which no microscope or imagingsystem is necessary but rather one or more of the light scatteringproperties are detected of a liquid or solid-phase sample through whichlight is scattered. These scattered light properties can be used todetermine the presence, absence or amount of analyte present in anyparticular sample.

The source of light in general need not be treated in any particularmanner (e.g., polarized, or laser or high intensity) but need only bedirected such that it allows scattered light from the particles to bedetected. Spatial filtering can be used to ensure reduction ofnon-specific light scatter. Such filtering can be supplemented by otherinstrumental components and sample chambers which reduce stray light.

The direct light can be polychromatic or monochromatic, steady-state orpulsed, and coherent or not coherent light. It need not be polarized andcan be generated from a low power light source such as a Light EmittingDiode (LED), or 12 watt filament light bulb and the like. The light isnot evanescent light, as described by Stimpson, supra. The light isdirected at a sample which may contain the particles at an angle suchthat the direct light itself will not be observed by the detector unlessit is scattered by the particles. The method and apparatus differs fromthat of Swope, supra in that such scatter can be observed by eye withinthe critical angle, preferably within the angle of illumination. It can,however, also be detected at greater than the critical angle and outsideof the intensity envelope of the forward direction of scattered light.When used with an imaging apparatus, e.g., a microscope, the presentmethod preferentially uses a detector perpendicular to the plane of thesample.

Unlike the diagnostic art that has been described in the “Background ofthe Invention”, Applicant has found that specific types of particles canbe detected and measured to very low concentrations, to a high degree ofspecificity, and across wide concentration ranges with easier to use andless costly methods and apparatus. The invention provides for detectionof analytes with greater ease of use, sensitivity, sensitivity,specificity, and is less costly than known methods of analyte detection.

Applicant has determined by methods of theoretical modeling and physicalexperimentation, that coated metal-like particles have similar lightscattering properties as compared to uncoated metal-like particles, bothof which have superior light scattering properties as compared tonon-metal-like particles. By “metal-like” particles is meant anyparticle or particle-like substance that is composed of metal, metalcompounds, metal oxides, semiconductor(SC), superconductor, or aparticle that is composed of a mixed composition containing at least0.1% by weight of metal, metal compound, metal oxide, semiconductor, orsuperconductor material. By “coated” particle is meant a particle has onit's surface a layer of additional material. The layer is there tochemically stabilize the particle in different sample environments,and/or to bind specific analytes by molecular recognition means. Suchcoatings are for example, inorganic and/organic compounds, polymers,proteins, peptides, hormones, antibodies, nucleic acids, receptors, andthe like. By “non-metal-like” particles is meant particles that are notcomposed of metal, metal compounds, superconductor, metal oxides,semiconductor, or mixed compositions that are not composed of at least0.1% by weight of metal, metal compound, metal oxide, superconductor, orsemiconductor material.

Applicant has also determined the following: (1) one or more analytes ina sample can be detected and measured by detection and/or measurement ofone or more of the specific light scattering properties of metal-likeparticles. These light scattering properties include the intensity,wavelength, color, polarization, angular dependence, and the RIFSLIW(rotational individual fluctuations in the scattered light intensityand/or wavelengths) of the scattered light. One or more of theseproperties of particle scattered light can be used to provideinformation regarding the analytes in the sample; (2) by varying thesize, and/or shape and/or composition of a metal-like particle invarious combinations, one or more of the light scattering properties canbe adjusted to generate more easily detectable and measurable lightscattering signals; (3) illumination and detection of the metal-likeparticles of certain size, shape, and composition with DLASLPD providesa highly sensitive and easy to use method to detect and measuremetal-like particles by their light scattering properties. The methodprovides for single particle detection with easy to use and inexpensiveapparatus means; (4) the DLASLPD methods can be used with particlecounting and/or integrated light intensity measurements to provide fordetection and measurement of the particles across wide concentrationranges; (5) the use of refractive index enhancement methods provides forenhancement of a particle's light scattering properties, and/ordecreases in non-specific light background; (6) the use of DLASLPD videocontrast enhancement methods can provide for more sensitive detection inmany different types of samples and diagnostic assay formats; (7) forsensitive detection of analytes in a small solid-phase area such ascommonly used in microarray and array chip formats, certain types ofmetal-like particles are more preferred to use than others. Metal-likeparticles in microarray and array chip formats can be most easily andinexpensively detected by using DLASLPD methods. Such particles in theseformats can also be detected by methods of laser scanning confocalmicroscopy, brightfield or epi-polarization microscopy, and reflectioncontrast and differential interference contrast microscopy. Howeverthese methods and apparatus are not as easy to use and inexpensive asdetection by DLASLPD methods and apparatus; and (8) useful apparatus andparticle types for specific test kits can be constructed. Thesedifferent test kits, and associated apparatus are useful forapplications to consumer use, portable field use, point of careapplications such as doctor's offices, clinics, emergency rooms and thelike, research laboratories, and centralized high throughput testing.The above aspects of the present invention provide for detection of oneor more analytes in many different types of samples and diagnostic assayformats.

As will be discussed in more detail below, there are many variations ofthe type of particle, and of the light source and light detectionmechanisms. In addition, many variations can be made on the types ofparticles used.

In preferred embodiments, the particle has a size, composition and shapesuitable for producing specific light scattering signal(s) of specificwavelength(s), color(s), polarization, angular dependence, and RIFSLIWof the scattered light that is detectable by eye or photodetector means;the detection includes the methods of counting the particles, and/ormeasurement of the intensity of scattered light as a measurement of theconcentration of the particles; the particle is formed from metal-likematerials or is formed from a mixed composition including non-metal-likematerials, the particles are spherical, oval, or asymmetrical (byasymmetrical is meant not roughly spherical in shape); the particles arecoated with binding agents, polymers, reactive chemical groups, basematerial molecules, inorganic and organic compounds; the change inscattered light properties when two or more particles are brought intoclose contact with each other in assay formats is used; particlereagents comprised of metal-like material and coated with base materialmolecules adapted to bind to a binding agent are used; assay formatswherein two or more particles are brought sufficiently close together sothat the light scattering property of the two or more particles can beresolved from single particles is used; assay formats wherein two ormore particles that are held in close proximity to one another arecaused to be separated so that the light scattering property of any oneparticle is altered is used; assay formats wherein two or more particlesare linked together by one or more molecular interactions such that whenthe molecular interaction holding the particles together is disrupted,one or more particles are released from the molecular interaction isused; assay formats wherein the amplified detection of analytes isaccomplished by cross-linking two or more particles together usingchemical or biological cross-linking agents is used; the particles arecomposed of additional materials to allow them to be oriented in anelectric, magnetic or related electromagnetic field (EMF); the particlesare attached to other particles of magnetic or ferro-electricproperties; and the illuminating light beam has a wavelength selected toreduce background as compared to other wavelengths.

In other embodiments, the illumination light is steady-state or pulsed;the illumination light is coherent or not coherent; the illuminationlight is polarized or unpolarized; two or more different wavelengthseither from the same light source or from two or more different lightsources are used to illuminate the sample, and the scattered lightsignals are detected.

In other embodiments, the method involves using a plurality of differentparticles each having one or more scattered light properties which canbe detected by eye or photodetector means; and/or a plurality ofdifferent wavelengths of light are used in the illumination or detectionstep; refractive index enhancement methods are used to reducenon-specific light background; the detector is placed at angles outsideof the envelope of the forward direction of sample and light beamscattered light; spatial filtering methods are used, optical filterssuch as cutoff and/or narrow band pass filters are used in the detectingstep to reduce non-specific background light.

In yet other embodiments, the particle is increased in size byautometallography prior to detection; the illuminating light beam lacksinfra-red radiation; the analyte is present in serum; the particles arereleased into solution prior to detection; the particles areconcentrated into a small volume or solid-phase area prior to detection;the particles are detected by time-dependent binding to a surface orflowing the particles pass a detector or set of detectors; multipleanalytes are detected on a solid-phase in a microarray; the microarrayis covered with liquid or is dry; single or multiple analytes aredetected on a cell surface, cell lysate, or chromosome preparation; theilluminating light beam is polychromatic white light or monochromaticlight; the analyte is present in solution or solid-phase, or is presenton a microscope slide, or a microtiter plate or another plasticcontainer; the particle is a gold or silver particle, having a sizebetween 1 and 500 nm, preferably between 10 and 200 nm; the detectingstep does not include amplification of the light scattered by electronicmeans; and the illuminating light beam is directed toward the particleby a prism or other light guiding systems.

In addition, the detection may include observing the particle through atleast a ×10 objective; the method of DLASLPD video contrast enhancementis used; fiber optic illumination and detection is used; bright-field,laser confocal scanning, reflection contrast or differentialinterference contrast microscopy detection methods are used; thedetection and purification of combinatorial synthesized molecules isperformed; particles and/or specialized coatings are used as solid-phasesynthetic supports for combinatorial or other synthesized molecules;specially designed sample chambers are used; antireflective coatings onoptical components and sample chambers are used; apparatus for fielduse, doctor's office, clinics and hospital care units are used; andspecific particle types are provided in appropriate test kits.

The high sensitivity and ease of use of the signal generation anddetection system of the present invention means that one skilled in theart can by inexpensive means, detect and measure one or more analytes ina sample to extremely low concentrations without need of signal (label)or target analyte molecule amplification methods.

The wide range of specific light scattering signals from differentparticle types in the present invention means that one skilled in theart can detect and measure to a high degree of specificity one or moreanalytes in a sample.

The high optical resolvability of two or more different particles typesin the present invention means that very simple multi-analyte detection(i.e. two or more different analytes) in a sample is possible withoutthe need for complex apparatus.

Those in the art will recognize that applicant has discovered novelmethods and apparatus with broad utility. The present invention can beapplied in one form or another to most situations where it is desirableto use a signal generation and detection system as part of an assaysystem to quantitate and/or detect the presence or absence of ananalyte. Such analytes include industrial and pharmaceutical compoundsof all types, proteins, peptides, hormones, nucleic acids, lipids, andcarbohydrates, as well as biological cells and organisms of all kinds.One or another mode of practice of this invention can be adapted to mostassay formats which are commonly used in diagnostic assays of all kinds.For example, these include heterogeneous and homogeneous assay formatswhich are of the sandwich type, aggregation type, indirect or direct andthe like. Sample types can be liquid-phase, solid-phase, or mixed phase.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments, and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings

FIG. 1 illustrates illumination of a sample from below. L is a lens; Dis the diameter of the lens; 0 is the area on surface S being detected;C is the cone showing the angles at which L collects light; LB is theilluminating light beam.

FIG. 2 illustrates the collecting angle of a lens. D is the diameter ofthe lens; f is the focal length; O is the area detected; θ_(H) is theplanar half angle of the collection cone.

FIG. 3 is a diagram which defines angles used to describe reflection andrefraction at a surface. S is the surface; ni and nt are the refractiveindices of incident medium and surface medium respectively; RFRB andRFLB are the refracted and reflected light beams respectively; IB is theincident light beam; θi, θr, and θt are the angles of incidence,reflection, and refraction of the light beam.

FIGS. 4A, 4B, and 4C are light reflection graphs for n_(i)<n_(t) takenfrom various texts.

FIGS. 5A, and 5B are light reflection graphs for n_(i)>n_(t) taken fromvarious texts.

FIG. 6 illustrates the refraction and reflection involved in theillumination of particles on a dry surface in air.

FIG. 7 is a graph of plot of θi2 vs. θi1 for n₂=1.5+n₃=1 (see FIG. 6).

FIG. 8 illustrates an angular distribution of light scattered by surfaceartifacts and particles; the dashed lines represent light scattered bythe particle; the solid line with an arrow is incident white light beam,and one beam of scattered light by surface artifacts, and the circle isan intensity envelope for the light scattered in the forward direction.

FIG. 9, Shows a sample in a thin film of water that is on a microscopeslide and covered with a cover glass. The illuminating beam encountersfour media interfaces; S1 (air to glass); S2 (glass to water); S3 (waterto glass); S4 (glass to air). The particles are at 0 on surface S2 orfreely moving above surface S2. Incident light strikes surface S1.

FIG. 10 shows illumination of a sample from above. L is the lens; C isthe collection cone.

FIG. 11 shows illumination using a prism arrangement (illumination frombelow). S1 is surface of prism where light is incident; S2 and S3 arethe bottom and top surface of a plastic piece substrate respectively.

FIGS. 12A, 12B, 12C, 12D, and 12E represent a pore prism (12A), an,equilateral prism (12B), a home made prism (12C and 12D), and a planeconvex lens, respectively.

FIG. 13 shows an illuminating light beam as viewed with a rhodamineplastic block.

FIG. 14 shows a surface and associated planes for descriptive purposesfor FIG. 15; S1 is solid substrate optically or not opticallytransmissive; SP1 is the 3-dimensional space above the plane of surfaceS1; SP2 is the 3-dimensional space below plane of surface S1. LightScattering particles or material is in the SP1 plane at or near thesurface S1.

FIG. 15 summarizes the different methods of DLASLPD illumination anddetection.

FIG. 16 shows the experimentally measured scattered light intensityversus incident wavelength spectrum for coated and uncoated 100 nmdiameter gold particles.

FIGS. 17, 18, and 19 show various sample chamber designs to reduce thelevel of non-specific light background. These sample chambers can testboth liquid and immobilized samples. For FIG. 17, S1 is the surfacewhere the light beam impinges on the sample chamber; S2 is the surfacethat contains the light scattering material(for immobilized samples). S3is another beveled surface; the surfaces S1 and S3 are beveled at anglesof from about 20 degrees to 70 degrees depending on the angle ofillumination, the face of surface S1 should be angled so that the lightbeam strikes S1 at 0 degrees with respect to the perpendicular; S4 is aoptically transmissive surface with or without an opening; S5 is theopposite side of the surface S2. If the chamber is enclosed(i.e. S4 issolid with no opening), a small opening is placed at one of the surfacesfor the introduction of sample and washing if required.

FIG. 18 is similarly designed as FIG. 17 except the beveled sides arereplaced with curved sides. Everything else is the same as the design ofFIG. 17.

FIG. 19, S1 is a flat beveled optically transmissive surface where thelight beam impinges on the sample chamber. The face of surface S1 shouldbe angled so that the light beam strikes S1 at 0 degrees; S2 is thesurface that contains the light scattering material if the material isimmobilized; S3 is another curved or beveled surface. S4 is an opticallytransmissive surface for a sample chamber that is enclosed;Alternatively, S4 has an opening of various size and shape forintroduction and washing of sample and detection; S5 is the oppositeside of the surface S2. If the chamber is enclosed, then a small openingis required at one of the surfaces for the introduction and/or washingof sample.

FIG. 20 shows a coordinate system that is used to describe theinteraction of particles with polarized light. Light travels along y andis polarized in the z direction. D is the detector of scattered lightintensity. λ is the direction of observation. θ and φ are the core angleand polar angle respectively.

FIG. 21 shows a schematic of an instrument for analysis of liquidsamples. Light from a filament or discharge lamp is focused by a lenssystem, represented by lens L1, onto the entrance slit of amonochromator; monochromatic light which exits the monochromator iscollected by lens L2 and focused by lens L3 onto the center of thetransparent sample cuvet (ST); the sample cuvet contains a liquidsolution of fluorescent molecules or suspension of light scatteringparticles; the sample cuvet is angled or inclined so that lightreflected from its walls is deflected downward and away from thephotodetector; light scattered or emitted by the sample is collected bythe lens L4 which forms at the plane M a magnified image of the samplecuvet and liquid contents; in the plane M is positioned a small aperturewhich selectively allows light emitted or scattered by the liquid at thecenter of the cuvet to reach the photodetector but blocks thephotodetector from light reflected or scattered from the side walls ofthe sample cuvet; the magnified image at M of the center of the liquidcontents is displaced from the optic axis of lens L4 by refractive indexeffects of the wall of the inclined sample cuvet through which theemitted or scattered light is detected; the photodetector and apertureare positioned to one side of the optic axis of lens L4 so that thedisplaced image of the liquid center falls on the aperture andphotodetector; holders for introducing optical filters and/or polarizersinto the illuminating light and scattered light paths are provided (H1and H2); a light shutter is positioned in front of the photodetector. Ifthe photodetector has a small light sensitive area, a lens L5 is used tofocus the light crossing the aperture at M unto the light sensitivearea. If monochromatic light is not required, the monochromator can beeasily removed and light from the filament or discharge lamp can bedelivered directly by lenses L1, L2 and L3 to the center of the samplecontainer.

FIGS. 22, 23, and 24 outline methods for using light scatteringparticles and specific DLASLPD methods, which leads to specific testkits and apparatus.

FIG. 25 outlines an apparatus and assay development process.

FIGS. 26 A, B, C, D, E, and F show the calculated scattered lightintensity versus incident wavelength profiles for spherical 10 nmdiameter gold, silver, aluminum, copper, selenium, and polystyreneparticles respectively. L_(k) is the wavelength; C_(sca) is the lightscattering cross section.

FIGS. 27 A, B, C, D, E, and F show the calculated scattered lightintensity versus wavelength of incident light for gold particles ofvarious sizes. A, B, C, D, E, and F correspond to spherical goldparticles of 10, 20, 40, 60, 80, and 100 nm in diameter respectively.REL CSCA is the relative light scattering cross section; WAVE,NM is thewavelength.

FIG. 28 is a diagram of a spherical coated particle. (1) is a coat ofpolymer, binding agent, or other substance on the surface of theparticle; (2) is the core particle.

FIGS. 29 A, B, and C show diagrams of MLSP (Manipulatable LightScattering Particle) mixed composition particles. A(1) is a coremagnetic or ferroelectric material coated with (2) the desired lightscattering material; B shows (4) a light scattering material core coatedwith (3) magnetic or ferroelectric material; C shows a mixture of (5)light scattering material with (6) magnetic or ferroelectric material.

FIGS. 30 A, B, and C show dimer, tetramer, and higher order particleconstructs respectively for orientable MLSP particles. (1) are lightscattering detectable particles and (2) are magnetic or ferroelectricparticles. The line (3) is the linkage chemical, ionic, or other thatbinds the particles together in the multi-particle construct.

The following abbreviations are used herein.

E-EMR—Emitted Electromagnetic Radiation

I-EMR—Incident Electromagnetic Radiation

EMF—Electromagnetic Field

SC—Semiconductor

Sec—Second

Q_(f)—Fluorescence Quantum Yield

I_(abs)—Incident Light Absorption (Photons absorbed per sec)

I₀—Incident Light Intensity (Photons per sec)

M—Molarity (Moles per liter)

ml—Milliliter

mM—Millimolar

g—gram

mg—milligram

mm—millimeter

μl—microliter

pI—Isoelectric point

E or e—Molar Decadic Extinction Coefficient (M⁻¹ cm⁻¹)

C—Molar Concentration (M)

X—Optical Path Length (cm)

I_(f)—Fluorescence Intensity (Photons per sec)

S_(eff)—Scattering efficiency of a particle

C_(abs)—Absorption Cross Section (cm²)

A_(CSR)—Ratio of Particle Absorbance Cross Section Over the ParticlePhysical Cross Section Area

C_(sca)—Scattering Cross Section (cm²)

S_(CSR)—Ratio of Particle Scattering Cross Section Over the ParticlePhysical Cross Section Area

a—Radius of a Particle

C_(ext)—Scattering Extinction Cross Section of Particle (cm²

I—Photons per sec which exit a solution after passing through a solutionthickness X

N—Particle Concentration (particles/cm³)

t—Turbidity of Suspension

I_(s)—Scattering Intensity (Photons/sec)

n₂—Refractive Index of Material

n₂Rel—Real Component of n₂

n₂Im—Imaginary Component of n₂

n₁—Refractive Index of Medium

m—The ratio of Refractive Index of Particle Material to Refractive Indexof Medium

l₀—Incident Light Wavelength (nm)

RI—Refractive Index Factor

Refmed—Refractive Index of Medium (n₁)

e_(m)—Dielectric constant of medium

n_(m)—Refractive index of medium

a—Determines the polarizability of the coated particle

nm—nanometer

cm—centimeter

μ—micron

DESCRIPTION OF THE INVENTION

This invention features a method for the detection and measurement ofone or more analytes in a sample. The method is based on the use ofspecific types of particles of certain composition, size, and shape andthe detection and/or measurement of one or more of the particle's lightscattering properties.

The present invention is easier to use, has greater sensitivity andspecificity, and is capable of detection and measurement across a widerconcentration range of analyte(s) than was previously possible. Thepresent invention has many advantages over the use of the methods ofsignal and target analyte amplification (e.g. chemiluminescence andPCR), fluorescence labels and fluorescence methods, and previousparticle-based assays and light scattering methods. The method isversatile and has broad application to the field of diagnostics as wellas other fields. The method can be used in most, if not all standardbinding-pair type assays such as immunoassay and nucleic acid assays andthe like for samples in the liquid-phase, mixed-phase, solid-phase, andsolid-phase microarray assay formats.

Rather than illustrate the broad utility by explicit illustrations ofeach specific practice of a particular form of the invention, applicantdescribes the key elements and considerations for one of average skillin the art to practice this invention to fit most if not all analytedetection needs. Such practice leads to specific apparatus and testkits.

The disclosure presented herein enables one of average skill in the artto practice the present invention in many different forms to achieve adesired analyte or particle detection capability to suit most if not allsample types, analyte types, diagnostic assay format types, andapparatus types. The present invention is so versatile that it can bepracticed to detect one or more analytes in the field (away from a lab),or in a small medical or analytical lab, at the bedside, emergencyrooms, specialized hospital care units (such as cardiac care, intensivecare, trauma unit and the like), a research lab, or the capability toprocess many samples a day. Different types of inexpensive apparatus andtest kits can be made by practice of the invention in one form oranother to suit a specific analytic diagnostic need.

There are several aspects of the invention which when practiced invarious combinations with each other define the analyte detectioncapabilities for a specific practice of the invention. Two of theseaspects are (1) the use of specific particle types that possess highlymeasurable and detectable light scattering properties in a defined assayformat and sample type, and (2) use of specific particle types with apreferred method of DLASLPD illumination and detection. In certainapplications, refractive index enhancement methods and DLASLPD videocontrast enhancement methods are also used.

Determination of Useful Light Scattering Properties of Metal-likeParticles

The following provides information helpful to fully understand theclaimed invention. These formulae are useful in practice andoptimization of the invention, but are not admitted to be prior art tothe claims.

In the development of the novel signal generation and detection systemfor the detection of analytes of the present invention, we found ituseful to develop new formulae which allowed us to evaluate variouslight scattering attributes of different particle types in terms offluorescence parameters. This allowed us to study ε, Q_(f), fluorescenceand excitation spectra, dependence of the emitted light intensity on theangle of observation, and state of polarization of the emitted light(these are defined below). These novel formulae allows one of skill inthe art to select the specific particle parameters such as composition,size, and shape to embody desirable light scattering property(s) thatcan be detected and measured when used in diagnostic assays or any otherapplication. Equations 1 through 7 are presented as backgroundinformation so the reader will understand the new formulations ofEquations 8 through 15. It should not be taken as an admission that anyof the formulae or light scattering parameters described is prior art tothe claims.

Applicant developed an analytical method based on certain modificationsof the art known light scattering theories of Rayleigh and Mie toevaluate many different types of particles with different parameters ofsize, shape, composition, and homogeneity to determine what specificconfigurations of particle parameters result in desirable lightscattering signals that are easily detected and measured in analyticaland diagnostic assays.

Definitions of Fluorescence Parameters

For fluorescent materials, the fluorescence intensity is determined bythe product of the number of photons which are absorbed per second andthe fraction of absorbed photons that are re-emitted as light (theQ_(f)) as shown by equation 1.

I _(abs)(λ)=2.303 I _(o)(λ) e(λ)Cx  (1)

where I_(o)(λ) is the incident light intensity (photons/sec) wavelengthλ, I(λ) is the molar decadic extinction coefficient in units of M⁻¹ cm⁻¹at wavelength λ and C is the molar concentration (in units of M) of thefluorophore and x is the optical path length in cm.

The integrated fluorescence intensity I(λ_(f)) (sum of photons emittedin all direction per sec) at the emission wavelength λ_(f) andexcitation wavelength λ_(e) is given by (for low fluorophoreconcentrations)

I(λ _(f))=2.303 I _(o)(λ_(e))Q _(f)(λ_(f))e(λ_(f))Cx  (2)

The assessment of the usefulness of a fluorescent compound in an assayapplication in terms of the listed parameters is a well known procedure.Use of fluorescent molecules and fluorescent techniques is limited bythe photostability of the fluorescent molecule, and the ability todetect the specific fluorescence emission signal in samples with highlevels of non-specific fluorescence, phosphorescence, and scatteredlight. For sensitive detection of fluorescent molecules or otherfluorescent substances such as particles composed of fluorescent dyemolecules, more sophisticated instrumentation is required.

Definitions of Light Scattering Parameters

Absorption Cross Section (C_(abs)) of a Particle

Consider a particle that is illuminated by a monochromatic beam of lightof wavelength λ. The absorption cross section C_(abs) of the particle isdefined as an area (usually expressed in units of cm² or μ²) surroundingthe particle, such that any photon falling on this area will beirreversibly absorbed by the particle. The value of C_(abs) depends onthe particle size, composition, shape and homogeneity. It also dependson the wavelength of light and a plot of Cabs vs. wavelength gives thepure absorption profile of the particle. The Cabs vs. wavelength profilefor any spherical particle with a homogeneous composition can becalculated with Rayleigh or Mie theory. In our terminology, C_(abs) isrelated to irreversible light absorption. The nature of C_(abs) can bebetter understood by reference to the section below where we define theextinction cross section C_(ext).

Relative Absorption Cross Section A_(csr)

The relative absorption cross section A_(csr) is defined as the ratio ofthe particle's C_(abs) divided by the physical cross sectional area ofthe particle πa² where a is the radius of the particle, i.e.,A_(csr)=C_(abs)/πa². A_(csr) provides a measure of the particle'sability to irreversibly absorb photons falling on the area surroundingthe particle. A_(csr) can have values in the range of 0 to 6 dependingon the composition, shape, and size of particle and the wavelength oflight. A value greater than one means that a particle can reach beyondits physical dimensions to attract photons to it and absorb them. In thephysical literature, A_(csr) is called the absorption efficiency factorof the particle. This nomenclature is misleading since A_(csr) can havevalues greater than 1, uncharacteristic of an efficiency.

Light Scattering Cross Section (C_(sca)) of a Particle

There is a finite probability that a photon of light absorbed (absorbedhere includes reversible and irreversible absorption) by a scatteringparticle is re-emitted at the same wavelength as the absorbed photon(quantum mechanical point of view). The re-emitted photon can be emittedin directions different from the direction of the incident photon. Thatis, the incident photons are scattered by absorption and re-emission.The scattering cross section of a particle (C_(sca)) at the incidentwavelength is defined as an area surrounding the particle such thatevery photon which falls on that area is scattered (that is absorbed andthen re-emitted in the quantum mechanical view). C_(sca) is usuallyexpressed in units of cm² or μ² and depends on the composition, shape,size and homogeneity of the particle and the wavelength. The lightscattering profile C_(sca) versus wavelength can be calculated for anyspherical particle of homogeneous composition using Rayleigh or Mietheory.

Ratio of C_(sca) to Physical or Geometric Cross Sectional Area of aParticle (S_(csr))

The ratio of the particle's C_(sca) divided by the physical orgeometrical cross sectional area of the particle πa², (where a is thespherical radius of the particle) provides a measure of the particle'sability to attract, absorb, and reemit photons from the area surroundingthe particle). That is S_(csr)=C_(sca)/πa². In the physical literature,S_(csr) is called the scattering efficiency factor.

Experimental and theoretical results show that the value of S_(csr) canbe in the range of 1 to 5 or greater depending on particle composition,shape, homogeneity and size and wavelength of light. A S_(csr) valuegreater than one means that a particle can reach beyond its physicaldimensions to attract photons to it and absorb and then re-emit them.This is possible because an electrical interaction of the particle withthe electromagnetic wave of the photon can occur at distances largerthan the radius of the particle. In general, S_(csr) increases withparticle size. For small particles (less than ˜40 nm) the S_(csr) isless than one while for larger particles Scsr equals greater than oneand can reach a value of five for the larger particles.

Extinction Cross Section (C_(ext)) of a Particle

The extinction cross section C_(ext) of a light scattering particle isdefined as the sum of the scattering cross section (C_(sca)) andabsorption cross section (C_(abs)) of the particle.

C _(ext) =C _(sca) +C _(abs)  (3)

C_(ext) is usually expressed in terms of cm² or μ².

The extinction cross section C_(ext) of any particle can be readilymeasured at any given wavelength in a regular absorption spectrometer.Let I_(o) (photons/sec) be the intensity of a light beam falling onsuspension of particles which are at a concentration of N particles/cm³.X(cm) is the thickness of the solution and I (photons/sec) is the amountof light which exits the solution after traversing the distance x. Theintensities are related to C_(ext) by the expression:

I _((λ)) =I _(o(λ)) e ^(−N) C _(ext(λ)) ^(x)  (4)

This expression shows explicitly the parameters depend on λ. It isassumed that the photodetector is positioned such that it does notdetect scattered light.

When the particles are pure scatterers, that is, do not irreversiblyabsorb any light, then c_(ext)=C_(sca) and the above equation is writtenas $\begin{matrix}{I = {I_{o}^{{- {NC}_{sca}}x}}} & (5) \\{\text{~~} = {I_{o}^{{- \tau}\quad x}}} & (6)\end{matrix}$

where t=NC_(sca) is the turbidity of the suspension.

Molar Decadic Extinction Coefficient

In the field of chemistry, the strength with which a substance insolution absorbs light at a given wavelength is expressed in terms ofthe molar decadic extinction e which has units of M⁻¹ cm⁻¹ (M stands formoles/liter). This coefficient is related to the experimentallydetermined absorbance by the expression

A _((λ)) =e _((λ)) Cx  (7)

Applicant Developed Formulae for Studying Particle Light ScatteringParameters

Applicant now briefly presents his own theoretical methods used. Oneskilled in the art can use the following methods to evaluate, modify,and adjust specific particle parameters of composition, size, shape, andhomogeneity to derive one or more desirable light scattering propertiesthat are easily detected and measured. Considerations must be made withregard to sample types, diagnostic formats, and limitations of apparatusmeans. For example, in one application, multi-analyte detection may beperformed on a solid-phase sample that contains a high non-specificlight background on a high throughput testing apparatus, while inanother application, single analyte detection in solution is performedin the doctors office.

Applicant's major interest was in optimizing particle types for use inanalytical and diagnostic assays. In most of these applications, theparticles must be coated with a macromolecular substance such aspolymer, protein, or the like to confer suitable chemical stability invarious mediums. This is known in the art. Applicant also places bindingagents such as antibodies, receptors, peptides, and the like on thesurface of the particle so that the coated particle can be used in ananalytic or diagnostic format. In some applications, the binding agentserves a dual function in that it stabilizes the particle in solutionand provides the specific recognition binding component to bind theanalyte. The coating of particles with proteins such as antibodies isknown in the art. However, applicant was interested in measuring one ormore specific parameters of the light scattering signals of differenttypes of particles which in some cases are of similar size and/or shapeand/or composition and it was not clear if such optical resolvability ofone or more of the specific light scattering properties of coatedparticles was possible.

Applicant determined by physical experimentation and theoreticalmodeling that the presence of thin coats of binding agents,non-optically absorbing polymers (in the visible region of thespectrum), or other materials on the particle surface does notnoticeably alter the light scattering properties specific for that typeof particle which is not coated with these types of materials. By “thincoat” is meant monolayer(s) of different amounts and compositions of theabove materials coated on the surface of the particle.

Applicant determined that a molar decadic extinction coefficient can bedetermined at any wavelength for a coated or uncoated particlesuspension by measuring its absorbance at that wavelength. The molardecadic extinction coefficient at that wavelength can then be calculatedwith Eq. (7) and the following expression to convert particleconcentration from N(particles/cm³) to C(M). M is moles/liter.

C(M)=1000 N(particles/cm³)/6.03×10²³  (8)

The molar decadic extinction coefficient can be related to theextinction cross section Cext (or vice versa) by the expression$\begin{matrix}{{ɛ\left( {M^{- 1}{cm}^{- 1}} \right)} = {{\left\lbrack {{C_{ext}\left( {{cm}^{2}/{particle}} \right)}\left( {6.03 \times 10^{23}} \right)} \right\rbrack/2.303} \times 1000}} & (9) \\{= {2.63 \times 10^{20}{C_{ext}\left( {{cm}^{2}/{particle}} \right)}\text{~~~~~~~~}}} & (10)\end{matrix}$

or $\begin{matrix}{{C_{ext}\left( {{cm}^{2}/{particle}} \right)} = {2.303\left( {M^{- 1}{cm}^{- 1}} \right) \times 1\text{,}{000/6.03} \times 10^{23}}} & (11) \\{\text{~~~~~~~~~~~~~} = {3.82 \times 10^{- 21}{ɛ\left( {M^{- 1}{cm}^{- 1}} \right)}}} & (12)\end{matrix}$

With Eq. (9) or (10) we can calculate ε from C_(ext).

As described previously, it is well known in the art that for particles,the extinction cross section (C_(ext)) is equal to the sum of thescattering cross section (C_(sca)) and the absorption cross section(C_(abs)). The extinction coefficient ε reflects the loss of photonsfrom the incident beam by irreversible absorption as well as byscattering (absorption and re-emission). Applicant has determined thatthe molar decadic extinction coefficient of a particle, evaluatedexperimentally or by calculation from the extinction cross section, canbe used to compare the absorption power of a particle with, for example,that of a fluorophore as shown later.

Light Scattering Efficiency (S_(eff))

Applicant determined that a light scattering efficiency S_(eff) can bedefined for a coated or uncoated particle, by analogy to fluorescenceefficiency Q_(f), as the fraction of photons absorbed (reversible plusirreversible absorption)by a particle that are re-emitted as scatteredlight. Mathematically, applicant defines the scattering efficiency bythe expression $\begin{matrix}\begin{matrix}{S_{eff} = {C_{sca}/C_{ext}}} \\{= {C_{sca}/\left( {C_{sca} + C_{abs}} \right)}}\end{matrix} & (13)\end{matrix}$

For particles which are pure scatters, that is, particles composed of amaterial which does not irreversibly absorb photons but only absorbs andre-emits photons, C_(abs) is equal to zero and S_(eff) is equal to one.Small polystyrene particles behave as pure light scatters in the visibleregion of the spectrum and S_(eff) is 1 for these particles. Forparticles composed of materials which reversibly and irreversibly absorbphotons, S_(eff) is less than one. Gold particles display the lattertype of behavior in the visible region of the spectrum.

Intensity of Light Scattered by a Particle

Applicant determined that the intensity of light scattered by a coatedor uncoated particle is determinable by the product of the number ofphotons which are absorbed (reversibly and irreversibly) per second andthe fraction of the absorbed photons that are re-emitted (quantummechanical point of view). Light scattering intensity measurements areusually done in dilute solutions where the amount of light absorbed,I_(abs) (photons absorbed per second) is given by

I _(abs) =I _(o)2.303εC x  (14)

I_(o) is the incident light intensity (photons/sec), ε is the molardecadic extinction coefficient of the particles in terms of M⁻¹cm⁻¹, Cis the molar concentration of the particles and x is the optical pathlength in cm.

Applicant further realizes that the total scattered light intensityI_(s), intensity integrated over all light scattering angles, is thengiven by the relation:

I _(s(λ))=2.303 I _(o(λ)) S _(eff(λ))ε_((λ)) (C) (x)  (15)

where I_(o(λ)) is the intensity of the incident light. This equation iscomparable to Eq. (2) for a fluorophore.

Note that when the expressions for ε and S_(eff) in terms of C_(sca) andC_(ext) are inserted in the above equation, the result shows that thescattered light intensity is directly proportional to and completelydetermined by the magnitude of the scattering cross section (C_(sca)).This means that the relative scattering intensities of differentparticles can be predicted from their scattering cross sections.

Specific Light Scattering Properties of Particles

Applicant now briefly summarizes some of the most important lightscattering properties that can be used to detect analytes in varioussample types using a variety of different assay formats. The measuredlight scattering properties that are detected are one or more of thefollowing: the intensity, the wavelength, the color, the polarization,the angular dependence, and the RIFSLIW (rotational individualfluctuations in the scattered light intensity and/or wavelengths) of thescattered light.

Coated and uncoated metal-like particles have similar light scatteringproperties and both have superior light scattering properties ascompared to non-metal-like particles. In addition, applicant hasdetermined that it is relatively easy to adjust the types of lightscattering properties in metal-like particles by varying in one form oranother, the size, shape, composition, and homogeneity such that thespecific light scattering attributes can be measured from the metal-likeparticle in various sample types.

Metal-like particles can be detected to extreme sensitivity. Theindividual particles are easily detected to the single particle limitusing DLASLPD illumination and detection methods with inexpensive andeasy to use apparatus.

One or more types of metal-like particles are detected in a sample bymeasuring their color under white light or similar broad bandillumination with DLASLPD type illumination and detection methods. Forexample, roughly spherical particles of gold (for example, coated withbinding agent, bound to analyte, released into solution or bound to asolid-phase) of 40, 60, and 80 nm diameters and a particle of silver ofabout 30 nm diameter can easily be detected and quantitated in a sampleby identifying each particle type by their respective unique scatteredlight color and/or measuring the intensity. This can be done on a solidphase such as a microtitier well or microarray chip, or in solution. Themeasurement in solution is more involved, because the particles are notspatially resolved as in the solid-phase format. For example, one candetect the different types of particles in solution by flowing thesolution past a series of detectors each set to measure a differentwavelength or color region of the spectrum and the intensity at thesedifferent wavelengths is measured. Alternatively, a series of differentwavelengths of illumination and/or detection can be used with or withoutthe flow system to detect the different particle types.

For solid-phase analytical applications, a very wide range ofconcentrations of metal-like particles is detectable by switching fromparticle counting to integrated light intensity measurements dependingon the concentration of particles. The particles can be detected fromvery low to very high particle densities per unit area.

In other assay applications, the particles which are bound to a solidsubstrate such as a bead, surface such as the bottom of a well, or thelike can be released into solution by adjusting the pH, ionic strength,or other liquid property. Higher refractive index liquids can be added,and the particle light scattering properties are measured in solution.Similarly, particles in solution can be concentrated by various meansinto a small volume or area prior to measuring the light scatteringproperties. Again, higher refractive index liquids can be added prior tothe measurement.

Both theoretical evaluation and physical experimentation indicate thatfor spherical particles of any composition up to about 120 nm indiameter and somewhat greater, a large proportion of the light scatteredby the particle is radiated outside the envelope of the forwarddirection of the scattered light (see FIG. 8). Applicant determined thatdetection and measurement of the scattered light at angles outside ofthe envelope of the forward direction of scattered light provides for asignificant decrease in non-specific scattered light from the light beamand other light scattering constituents and objects being detected. Thissignificantly increases the specific light scatteringsignal/non-specific light scattering ratio for many samples.

The intensity of light scattered by a particle in different directionsand the state of polarization of the scattered light depends on thewavelength and state of polarization of the incident light and on theparticle size, shape and homogeneity. Below we summarize some of themost important facts concerning the intensity and state of polarizationof light emitted in different directions by certain types of particles.

Smaller particles of spherical shape (smaller being about {fraction(1/20)} or smaller as compared to the wavelength of light) behave asisotropic dipole scatterers or emitters, that is, the light is highlypolarized. This is very different to fluorescent molecules which usuallybehave as linear dipole emitters. For example, such particles whenilluminated with unpolarized light, the light scattered in the directionφ=0, θ=90 (see FIG. 20) is one hundred percent linearly polarized (P=1).This property allows for the more specific and more sensitive detectionof analytes by measurement of the light scattering properties ascompared to fluorescent molecules in many different types of samples.

For even larger particles (>{fraction (1/20)} wavelength of light) thereare certain ranges of particle sizes where the degree of polarization oflight P decreases and becomes dependent on the wavelength as the sizeincreases. As the particles become very large the degree of polarizationapproaches 0 for the direction θ=0, φ=90°. There appears to be certainsize ranges where the change in polarization changes the most, that is,the slope of degree of polarization versus size is at a maximum. Thoseregions where the slope is changing are used in certain analyticapplications as for example, agglutination or aggregation types ofassays to detect and measure one or more analytes in a sample.

For larger spherical particles in certain size ranges, for example fromabout 200 nm to about 1.2 microns in diameter, the intensity of lightoscillates (for monochromatic incident light) between relative values of1 to 0 as the angle φ is changed from 90° to −90° for θ=0 with referenceto FIG. 20. That is, if one views the scattered light in the horizontalplane (θ=0), the light intensity oscillates from bright to dark as theeye is moved from θ=90° to θ=−90°. For illumination with white light,the light changes color as the eye is moved from θ=90° to θ=−90°. Thatis, the particles behave as diffraction gratings. Such light scatteringproperties are very useful to detect more specifically and to greatersensitivity one or more analytes in many different types of samples.

Small nonspherical particles behave somewhat as linear dipole scatterswith the absorption and emission moments along the long axis of theparticle. Applicant has observed the following under DLASLPDillumination and detection conditions in an ordinary light microscope.When the illuminating light is linearly polarized, the non-sphericalparticles flicker as they rotate. The particles long axis is oriented inthe direction of polarization and is at a minimum when the moment isperpendicular to this direction. In contrast, small spherical particlesdo not flicker when illuminated by polarized light. For nonsphericalparticles of certain compositions, the color of the scattered light(with white light illumination) changes with the degree of asymmetry. Asthe asymmetry is increased, the color shifts towards longer wavelengths.For example, asymmetric particles of silver were observed by applicantto change colors as the particles were rotating in solution when viewedwith an ordinary light microscope under DLASLPD like conditions. Thisproperty termed “RIFSLIW” by applicant (rotational individualfluctuations in the scattered light intensity and or wavelengths) isused in many different aspects of the current invention to morespecifically and more sensitively detect and or measure one or moreanalytes or particles in a sample.

Applicant has also determined that certain mixed compositions ofparticles made from metal-like materials, and non-metal-like andmetal-like materials provides for additional light scattering propertiesand/or additional physical properties. These properties include theability to manipulate the particles by applying an EMF field. Thisproperty of the particles can be used in many different ways with thepractice of one or more aspects of this invention. Applicant nowprovides further illustrative discussions of particle-dependent lightscattering properties and the use of these properties to detect one ormore analytes in a sample.

It will be useful to first describe the present invention in terms ofthe light scattering properties of homogeneous, spherical particles ofdifferent sizes and compositions. However, the basic aspects of theinvention apply as well to non-spherical particles as one in the art candetermine. In addition, it will be useful to describe the presentinvention in terms of the incident light wavelengths in the range 300 nmto 700 nm. However, the basic aspects of the invention apply as well toelectromagnetic radiation of essentially all wavelengths. By “light” ismeant ultraviolet, visible, near infrared, infrared, and microwavefrequencies of electromagnetic radiation. It will be further useful forthe description of the present invention to use polystyrene particles torepresent non-metal-like particles of various types. Othernon-metal-like particle types include those composed of glass and manyother polymeric compounds. Such particles have roughly similar lightscattering characteristics as compared to polystyrene particles.

The relative intensities of scattered light obtained from differentparticles irradiated with the same intensity and wavelengths of incidentlight can be directly compared by comparing their C_(sca')s. The higherthe C_(sca), the greater the scattering power (light scatteringintensity) of the particle. In the following sections we use the words“scattering power” to mean C_(sca) or scattered light intensity.

We have calculated the light scattering powers, in water, of smallspherical particles identical in size, and different in composition, forincident wavelengths over the wavelength ranges of 300 to 700 nanometers(nm) . In these calculations, we have used values of refractive indexvs. wavelength tabulated in standard handbooks for different bulkmaterials in vacuum.

For some particle compositions, the light scattering power decreasescontinuously from 300 to 700 nm while for other compositions thescattering power vs. wavelength profile shows peaks or bands. When thesepeaks or bands are in the visible region of the spectrum the lightscattered by the particles is colored when the incident light is white.

For illustrative purposes we show in some of the following tablesvarious comparisons of light scattering properties for different typesof 10 nm diameter particles. The general trends with regard to therelative magnitudes and wavelengths of scattered light that isdemonstrated for these 10 nm diameter particles is generally the samefor larger particles up to about 100 nm. For example, the calculationsof Table 1 were done with particles of ten nanometer diameter. However,for small particles( less than about {fraction (1/20)} wavelength of thelight) the light scattering intensity vs. wavelength profiles do notchange in shape as the particle size is increased as long as one remainsin the small particle limit. The apparent effect of increase in particlesize is to increase the amplitude of the profile graph. It is well knownin the art of theoretical physics and light scattering that thescattering power of small particles increases with the sixth power ofthe radius. One skilled in the art can calculate the relative scatteringpower of any small particle of diameter d from the value obtained forthe 10 nm diameter particle by multiplying the light scattering power ofthe 10 nm particle by (d/10)⁶ where d is in nm. This method can be usedby one skilled in the art to determine the usefulness of certainparticle sizes in various diagnostic assay applications where theintensity of the scattered light of a particle is used to detect thepresence of an analyte.

From our theoretical and physical experimentation we were surprised tofind that this general relationship does also apply to larger particlesoutside of the Rayleigh limit, that is, for particles with diameterslarger than about 30 nm.

Table 1 presents the calculated C_(sca) values(light scattering power)and their respective approximate wavelengths in the visible range wherethe particles scatter light most intensely. The data of Table 1 suggestthat metal-like particles are much more powerful light scatterers than,for example, polystyrene particles.

FIG. 26 shows selected calculated light scattering intensity vs.wavelength profiles for certain types of 10 nm spherical particles.Small particles composed of gold or silver exhibit very prominentscattering and absorption peaks in the visible wavelength region, whilecopper particles exhibit a small scattering and absorption peak in thisregion. The gold and silver scattered light maxima occur at about 530 nmand 380 nm respectively. Even at incident wavelengths far removed fromthe light scattering maxima, the light scattering power of the gold,silver, and other metal-like particles is much greater than that ofnon-metal-like polystyrene particles of similar size.

Table 2 presents the calculated light scattering power (C_(sca)) valuesfor metal-like particles and polystyrene(non-metal-like) of 10 nmdiameter when the incident (illumination) wavelength has been shifted tomuch longer wavelengths. In many different analytic and diagnosticassays, it is preferable to work at longer wavelengths. Table 2indicates that one skilled in the art may use illumination wavelengthsat much longer wavelengths and that the metal-like particles are farsuperior to non-metal-like particles as for example, polystyrene forapplications to analytical or diagnostic methods. For example, at anincident light wavelength of 700 nm, a wavelength far from the lightscattering maximum at 530 nm for gold particles, the data suggest that agold particle's scattered light intensity is about 220 times more than apolystyrene particle of similar size and shape. We have experimentallyobserved that indeed the light scattering power(intensity) of themetal-like particles is much greater than non-metal-like particlesacross the visible wavelengths of the spectrum.

These results indicate that metal-like particles have much greater lightscattering power than non-metal-like particles of comparable size andshape and are broadly applicable as analytical and diagnostic tracersfor use in most areas where it is desirable to use a signal generationand detection system. For example, in any assay designed to detect thepresence or absence of an analyte substance by detecting the scatteredlight from the particles.

TABLE 1 CALCULATED (C_(sca)) VALUES FOR TEN NANOMETER SPHERICALPARTICLES OF DIFFERENT COMPOSITION IN WATER Particle Wavelength MaximumComposition (nm)^((a)) C_(sca (cm) ²) Relative C_(sca) Polystyrene 3001.32 × 10⁻¹⁷ 1 Selenium 300  8.6 × 10⁻¹⁶ ˜65 Aluminum 300 1.95 × 10⁻¹⁵˜148 Copper 300  7.8 × 10⁻¹⁶ ˜59 Gold   530^((b)) 1.24 × 10⁻¹⁵ ˜94Silver   380^((b))  1.1 × 10⁻¹⁴ ˜833 ^((a))Incident wavelength used andat which maximum value occurs in the visible range of the EM spectrum(300-700 nm). ^((b))Some particles display peaks in certain regions. SeeFIG. 27.

TABLE 2 CALCULATED VALUES OF (C_(sca)) AT 700 NM ILLUMINATION FOR TENNANOMETER SPHERICAL PARTICLES OF DIFFERENT COMPOSITIONS IN WATERParticle Incident Maximum Relative Composition Wavelength (nm) C_(sca)(cm²) C_(sca) Polystyrene 700 ˜3.1 × 10⁻¹⁹ 1 Selenium 700 ˜1.4 × 10⁻¹⁷˜45 Aluminum 700  1.4 × 10⁻¹⁷ ˜45 Copper 700  4.7 × 10⁻¹⁷ ˜152 Gold 700  7 × 10⁻¹⁷ ˜225

Table 3 shows a comparison of the molar decadic extinction coefficients(ε) calculated and experimentally measured for spherical gold particlesof different diameter.

We calculated the ε values at the wavelength of maximum value using theexpressions previously described. Measured ε values were obtained bymeasuring the optical absorption in a standard spectrophotometer at thecalculated wavelength of maximum absorption. The agreement betweencalculated and experimentally measured ε values while not perfect, isquite good. The approximately two-fold differences observed between theobserved and calculated results may reflect inaccuracies in the stateddiameters of the gold particles. Details of the experimental methods aregiven in the Example Section.

TABLE 3 CALCULATED AND MEASURED MOLAR DECADIC EXTINCTION COEFFICIENT(S)AND WAVELENGTHS OF MAXIMUM ABSORPTION FOR GOLD PARTICLES OF DIFFERENTSIZE IN WATER CALCULATED^((b)) MEASURED^((b)) WAVELENGTH WAVELENGTHPARTICLE AT MAXIMUM AT MAXIMUM DIAMETER^(a,b) ε (M⁻¹ cm⁻¹) ABSORPTION ε(M⁻¹ cm⁻¹) ABSORPTION 10 nm 1.8 × 10⁸  ˜530 nm 2.3 × 10⁸  ˜525 nm 16 nm  9 × 10⁸  ˜530 nm 5.2 × 10⁸  ˜522 nm 20 nm 1.8 × 10⁹  ˜528 nm 1.2 ×10⁹  ˜525 nm 40 nm 1.6 × 10¹⁰ ˜532 nm 8.5 × 10⁹  ˜528 nm 60 nm 5.6 ×10¹⁰ ˜542 nm 2.6 × 10¹⁰ ˜547 nm 80 nm 1.1 × 10¹¹ ˜550 nm 5.8 × 10¹⁰ ˜555nm 100 nm  1.6 × 10¹¹ ˜574 nm 9.2 × 10¹⁰ ˜575 nm ^((a))Denotes exactdiameter of particle for calculating values. ^((b))Denotes approximatediameter of gold particles used for measurements. Actual diameters wereslightly higher or slightly lower than the diameters noted.

At visible wavelengths of incident light, the light scattering power(i.e., C_(sca)) of metal-like particles is much greater than for acomparable non-metal-like particle such as polystyrene. Anotherimportant distinction between the light scattering properties ofmetal-like and non-metal-like particles is that for metal-likeparticles, the profile of scattered light intensity versus incidentlight wavelength for metal-like particles of same composition butvarying size can be very different. This is in contrast tonon-metal-like particles where in the size ranges of about 10 nmdiameter to a few hundred nm diameter the profile is essentially thesame. These differences are extremely useful to more specifically andmore sensitively detect metal-like particles in various samples. Theincident wavelength at which maximum light scattering (C_(sca)) occursfor various diameter particles of silver, gold, copper, and aluminum arepresented in Table 4.

FIG. 16 shows the experimentally measured scattered light intensity vs.incident light wavelength profile for roughly spherical 100 nm diametergold particles coated with polyethylene compound (MW=20,000) and withoutthe polyethylene compound. The data show that the wavelength dependentlight scattering intensity properties of the coated and uncoated 100 nmdiameter gold particle are very similar.

FIG. 27 shows the calculated scattered light intensity versus incidentlight wavelength spectra profiles for spherical gold particles ofvarying diameter. The scattered light intensity peak wavelengths shiftto longer wavelengths as the size of the gold particles is increased. Wehave directly observed these light scattering properties for coated oruncoated gold particles of 40, 60, 80, 100 nm diameters and they appearas green, yellow-green, orange, and orange-red particles whenilluminated with a white light source in solution or in a lightmicroscope using DLASLPD illumination methods. Small spherical silverparticles appear blue. Thus, metal-like particles coated with varioustypes of binding agents can be used in numerous ways in analytic typeassays. The color properties of the scattered light of different typesof metal-like particles allows for multi-analyte visual detection. Forexample, spherical gold particles of 40, 60, 80, and 100 nm diameter and20 nm diameter silver particles, each coated with a different type ofbinding agent, can be used in the same sample to detect five differentanalytes in the sample. In one format, five different types of cellsurface receptors, or other surface constituents present on the cellsurface can be detected and visualized. Detection of the scattered lightcolor of the differently coated particles that are bound to the surfaceof the cell under DLASLPD conditions with a light microscope with whitelight illumination makes this possible. The number and types of analytesare identified by the number of green, yellow, orange, red, and blueparticles detected. Similarly, chromosome and genetic analysis such asin situ hybridization and the like can also be done using the method asdescribed above where the different types of metal-like particles areused as “chromosome paints” to identify different types of nucleic acidsequences, nucleic acid binding proteins, and other similar analytes inthe sample by the color of the scattered light of the different types ofmetal-like particles. These examples are provided as illustrativeexamples, and one skilled in the art will recognize that the color ofthe scattered light of different types of metal-like particles can beused in many different assay formats for single or multi-analytedetection.

Thus, adjusting the size of certain types of spherical metal-likeparticles is a useful method to increase their detectability in varioussamples by using the color and/or other properties of their scatteredlight. By using a white light source, two or more different types ofparticles are easily detectable to very low concentrations.

Table 5 shows that modest increases in gold particle size results in alarge increase in the light scattering power of the particle (theC_(sca)). The incident wavelength for the maximum C_(sca) is increasedsignificantly with particle size and the magnitude of scattered lightintensity is significantly increased. For example, the incidentwavelength for maximum C_(sca) is around 535 nm, 575 nm and 635 nm forgold particles 40 nm, 100 nm, and 140 nm in diameter, respectively. Whenilluminated with white light, the 40 nm gold particles strongly andpreferentially scatter the wavelengths around 535 nm and the particlesappear green, while the 100 nm particles appear orange-red and the 140nm particles appear red in color. This further shows that whenilluminated with white light, certain metal-like particles of identicalcomposition but different size can be distinguished from one another inthe same sample by the color of the scattered light. The relativemagnitude of the scattered light intensity can be measured and usedtogether with the color or wavelength dependence of the scattered lightto detect different particles in the same sample more specifically andsensitively, even in samples with high non-specific light backgrounds.

In contrast, for non-metal-like particles, these particles do notpossess these specific types of light scattering properties and thus, itis more difficult to detect the non-metal-particles in most types ofsample medium as compared to the metal-like particles.

TABLE 4 CALCULATED INCIDENT VISIBLE WAVELENGTH AT WHICH MAXIMUM C_(sca)IS OBSERVED IN WATER WAVELENGTH OF INCIDENT PARTICLE PARTICLE LIGHT ATWHICH MATERIAL DIAMETER MAXIMUM C_(sca) OCCURS Silver  10 nm ˜380 nm  40nm ˜400 nm 100 nm ˜475 nm Gold  10 nm ˜528 nm  40 nm ˜535 nm 100 nm ˜575nm 140 nm ˜635 nm Copper 100 nm ˜610 nm 150 nm ˜644 nm Aluminum 100 nm˜377 nm Selenium 130 nm ˜660 nm 200 nm ˜702 nm

TABLE 5 CALCULATED VALUES FOR LIGHT SCATTERING CHARACTERISTICS OFSPHERICAL GOLD PARTICLES OF DIFFERENT SIZES WAVELENGTH CALCULATEDPARTICLE AT MAXIMUM MAXIMUM RELATIVE DIAMETER C_(csa) C_(sca) SCATTERING(nm) (nm) (cm²) POWER 10 ˜528 1.26 × 10⁻¹⁵ 1 20 ˜525  8.4 × 10⁻¹⁴ 67.530 ˜530 1.03 × 10⁻¹² 817 40 ˜535   6 × 10⁻¹² 4.8 × 10³ 60 ˜545  6.3 ×10⁻¹¹   5 × 10⁴ 80 ˜555  2.3 × 10⁻¹⁰ 1.8 × 10⁵ 100 ˜575  4.6 × 10⁻¹⁰ 3.6× 10⁵ 120 ˜605  6.9 × 10⁻¹⁰ 5.5 × 10⁵ 140 ˜635  8.8 × 10⁻¹⁰   7 × 10⁵160 ˜665   1 × 10⁻⁹  7.9 × 10⁵ 200 ˜567  1.4 × 10⁻⁹  1.1 × 10⁶ 300 ˜670 2.9 × 10⁻⁹  2.3 × 10⁶ 600 ˜600 1.01 × 10⁻⁸    8 × 10⁶ 1,000 ˜620  2.5 ×10⁻⁸  1.8 × 10⁷ 1,000 ˜670  2.5 × 10⁻⁸  1.8 × 10⁷

The relative light scattering powers of particles of the same shape andsize, but of different composition, can be directly comparedexperimentally by comparing the light scattering intensities at rightangles to the path of the incident light. We experimentally compared therelative light scattering powers of gold and polystyrene particles ofsimilar size and shape, using a light scattering instrument we builtdesigned to measure scattered light at right angles to the path of theincident light and which is described elsewhere. Table 6 shows that theexperimentally measured scattering power of a particle composed of goldis much greater than the scattering power of a particle composed ofpolystyrene when both particle types are compared at the same incidentvisible wavelength. The experimentally measured values of Table 6 are afactor of two to three lower than the calculated values. A large part ofthis difference can be attributed to the approximately two-fold lowervalues obtained for the experimentally measured molar decadic extinctioncoefficients of gold particles relative to the calculated values (seeTable 3). In addition, there is a certain level of uncertainty inparticle sizes (e.g. for a polystyrene particle preparation 21 nm+1.5 nmthe actual size could be about 1.5 nm larger or smaller). Thisuncertainty makes the quantitative values less certain for both thepolystyrene and gold particles but does not change the basic conclusionconcerning the relative scattering powers. Even at the greatest level ofuncertainty, Table 6 indicates that at a minimum, the scattering powerof a gold particle is 100 to 200 times greater than that of apolystyrene particle of comparable size and shape.

Table 7 compares the relative light scattering power of spherical goldand polystyrene particles of similar size and shape, at differentwavelengths of visible light. Table 7 indicates that even atillumination wavelengths far away from the wavelength of maximum lightscattering intensity, the light scattering power of a gold particle ismuch greater than that of a polystyrene particle of comparable size andshape. These experimental results agree with our calculated results(seeTable 2).

Table 8 shows that the experimentally determined light scattering powerof spherical gold particles is much greater than comparable polystyreneparticles using white incandescent light illumination conditions.

Overall, the agreement between our calculated and experimentallydetermined results presented herein is quite good. This validates thecalculated results as well as the use of the calculation process foridentifying potentially useful particle materials and compositions andfor evaluating the utility of the light scattering properties of suchparticles. Most types of light sources which produce polychromaticand/or monochromatic light, steady-state and/or pulsed light, andcoherent or not coherent light can be used for illumination. Our resultsindicate that more specific and more intense light scattering signalscan be can be obtained from metal-like particles as compared tonon-metal-like particles of comparable size and shape. Our resultsindicate that the present invention provides a means to detect lesseramounts of particles, and to more specifically detect lesser and greateramounts of particles than was previously possible.

TABLE 6 CALCULATED AND MEASURED RELATIVE SCATTERING POWER IN WATER OFPOLYSTYRENE AND GOLD PARTICLES OF SIMILAR SIZE AND SHAPE AT THE INCIDENTWAVELENGTH AT WHICH MAXIMUM SCATTERING OCCURS FOR THE GOLD PARTICLESPARTICLE PARTICLE INCIDENT RELATIVE SCATTERING POWER COMPOSITIONSIZE^((b)) WAVELENGTH ^((a))CALCULATED MEASURED PST^((c))  21 ± 1.5 nm˜525 nm 1 1 Gold 19.8 ± <1.9 nm ˜525 nm ˜664 ˜220 PST  32 ± 1.3 nm ˜527nm 1 1 Gold 29.5 ± <3.5 nm ˜527 nm ˜663 ˜318 PST  41 ± 1.8 nm ˜530 nm 11 Gold 39.5 ± <6 nm   ˜530 nm ˜985 ˜461 PST  83 ± 2.7 nm ˜560 nm 1 1Gold 76.4 ± 15 nm   ˜560 nm ˜708 ˜211 ^((a))Calculated for perfectlyspherical particles ^((b))Manufacturer's measured particle sizes. Themanufactured particles are spherical in shape but not perfectlyspherical. This will have little effect on the quantitative aspects ofthis comparison. ^((c))PST—polystyrene

TABLE 7 MEASURED RELATIVE SCATTERING POWER IN WATER OF POLYSTYRENE ANDGOLD PARTICLES OF SIMILAR SIZE AND SHAPE AT INCIDENT WAVELENGTHS AWAYFROM THE GOLD PARTICLE ABSORPTION BAND RELATIVE SCATTERING POWER VISIBLE^((c))CORRECTED WAVELENGTH FOR PARTICLE PARTICLE INCIDENT OF MAXIMUMDIRECT PARTICLE COMPOSITION SIZE^((a)) WAVELENGTH SCATTERING COMPARISONSIZE PST^((b)) 41.1 ± 1.8 nm  ˜530 nm ˜300 nm 1 1 ˜460 nm 1 1 ˜400 nm 11 Gold 39.9 ± <6 nm   ˜530 nm ˜530 nm ˜377 ˜443 ˜460 nm ˜96 ˜113 ˜400 nm˜80 ˜94 PST  83 ± 2.7 nm ˜560 nm ˜300 nm 1 1 ˜430 nm 1 1 ˜380 nm 1 1Gold 76.4 ± <15 nm  ˜560 nm ˜560 nm ˜251 ˜412 ˜430 nm ˜36 ˜55 ˜380 nm˜27 ˜41 ^((a))Manufacturer's measured particle size^((b))PST—Polystyrene ^((c))Adjust the relative light scattering valuesfor the gold particles for any difference in size between the PST andgold particles by using the relationship that scattering power increasesas the sixth power of the radius. Although in these size ranges, this isnot quite accurate, the corrected figures are a good approximation.

TABLE 8 MEASURED RELATIVE SCATTERING POWER IN WATER OF WHITE LIGHTILLUMINATED POLYSTYRENE (PST) AND GOLD PARTICLES OF A SIMILAR SIZE ANDCOMPOSITION ^((c))RELATIVE RELATIVE SCATTERING SCATTERING POWER POWERADJUSTED AT SAME FOR ^((a))PARTICLE ^((b))PARTICLE CONCEN- PARTICLECOMPOSITION SIZE TRATION SIZE PST   21 nm 1 1 GOLD 19.8 nm ˜40 ˜58 PST  38 nm 1 1 GOLD 39.9 nm ˜212 ˜158 PST 37.9 nm 1 1 GOLD 39.9 nm ˜105 ˜77PST   59 nm 1 1 GOLD 59.6 nm ˜100 ˜94 PST   79 nm 1 1 GOLD 76.4 nm ˜108˜132 ^((a))Polystyrene particles obtained from interfacial Dynamics,Inc., Portland, Oregon or Duke Scientific, Inc., Palo Alto, CA. Whilethe gold particles were obtained from Goldmark Biologicals,Phillipsburg, N.J. a distributor for British Biocell LTD., Cardiff, UK.^((b))Manufacturers represented particle diameter. ^((c))Done using therelationship that scattering power increases approximately as the sixthpower of the particle radius.

Particle Light Generation Power Compared to Fluorescence

Fluorescence is currently being used in many assays designed to detectthe presence or absence of an analyte substance.

Fluorescein is one of the best understood and most widely usedfluorescent compounds. Many studies have been conducted with the purposeof detecting as few fluorescein molecules as possible. Fluorescein has ahigh molar decadic extinction coefficient (about 6×10⁴ M ⁻¹cm⁻¹) and hasa very high fluorescent quantum yield of about 0.8.

Table 9 compares the calculated signal generating power of certainparticles to fluorescein. Clearly, a single gold or silver particle is amuch more intense light source than a single fluorescence molecule.Under ideal conditions and using appropriate optical filters, a goodfluorimeter can detect fluorescein at a lower concentration of about10⁻¹⁰ M to 10⁻¹¹M. The comparison presented in Table 9 indicates thatthis same fluorimeter should be able to detect a lower concentration ofa 60 nm gold particle of around 10⁻¹⁵M−10⁻¹⁶ M. We have verified theseobservations experimentally.

Table 9 indicates that the total scattered light output from a single 60nm gold particle is equivalent to the output of about 350,000fluorescein molecules. While one fluorescein molecule cannot be directlyvisualized in the light microscope, we are able to directly visualizeindividual metal-like particles in many different types of samples andassay formats. The light is directed at the sample with such an angle sothat the light scattered from the particle is maximally visualized ormeasured by the eye or photodetector. This broadly applicable method ofillumination and detection as we have developed in one form or anotherfor use in analytic and diagnostic applications is called DLASLPD(direct light angled for scattered light of particle only to bedetected). These methods are described in greater detail elsewhere. Thisallows the detection of single particles and the quantitation of suchparticles by particle counting methods including image analysis, photoncorrelation spectroscopy, light microscopy and other methods. Incontrast, only very large particles of polystyrene can be seen in thelight microscope using DLASLPD techniques.

Table 10 presents results comparing the experimentally measured relativesignal generating power of fluorescein and gold particles of varioussizes using white light illumination. These results are similar to thosepresented in Table 8 and show that the light generation power of a goldparticle is much greater than a fluorescein molecule. For example, goldparticles of a diameter of 39.9 nm and 59.6 nm emit a light intensityequivalent to that given off by about 2×10⁴ and 2.3×10⁵ fluoresceinmolecules respectively, when illuminated with white light.

The scattered light emitted by gold particles illuminated with whitelight is composed of all of the wavelengths present in the incidentwhite light, but the efficiency of light scattering at any particularwavelength varies such that one or more bands of scattered lightwavelengths are scattered more intensely. The actual wavelengthcomposition and the scattered light wavelength versus scattered lightintensity profile obtained when white incident light is used, depends ona number of variables which include the type of light source used andthe method of light detection. The results of Table 10 were obtainedwith an incandescent light source or color temperature of 2,800° Kelvinand the light was passed through a simple filter to reduce the infraredcomponent before passing through the sample. The scattered lightintensity was measured with a standard photomultiplier tube. The resultsof Table 10 have not been corrected for phototube or light sourceproperties. Any such corrections would not affect the conclusionsdiscussed herein.

TABLE 9 CALCULATED RELATIVE SIGNAL GENERATING POWER OF FLUORESCEIN ANDSPHERICAL PARTICLES OF VARIOUS COMPOSITIONS AND SIZES NUMBER OFFLUORESCEIN MOLECULES NECESSARY TO MATCH THE PARTICLE TOTAL LIGHTPARTICLE PARTICLE VOLUME INTENSITY FROM COMPOSITION DIAMETER (micron)³ONE PARTICLE^((a)) Polystyrene  10 nm 5.23 × 10⁻⁷ ˜0.07  20 nm  4.2 ×10⁻⁶ ˜5  40 nm 3.35 × 10⁻⁵ ˜280  60 nm 1.13 × 10⁻⁴ ˜2800 100 nm 5.23 ×10⁻⁴ ˜42,000 Silver  10 nm 5.23 × 10⁻⁷ ˜46  20 nm  4.2 × 10⁻⁶ ˜3,500  40nm 3.35 × 10⁻⁵ ˜150,000  60 nm 1.13 × 10⁻⁴ ˜770,000 100 nm 5.23 × 10⁻⁴˜2,300,000 Gold  10 nm 5.23 × 10⁻⁷ ˜7  20 nm  4.2 × 10⁻⁶ ˜455  40 nm3.35 × 10⁻⁵ ˜35,000  60 nm 1.13 × 10⁻⁴ ˜350,000 100 nm 5.23 × 10⁻⁴˜3,100,000 ^((a))Fluorescein and the particle are illuminated with thesame light intensity at wavelengths which generate the maximumfluorescent or light scattering signal. For polystyrene the incidentlight wavelength used was 300 nm, while the incident light wavelengthused for each gold or silver particle was the wavelength at maximumC_(sca) for the various sizes.

Results from measurement of the relative signal generating powers offluorescein and roughly spherical gold particles of different size whenilluminated with incident monochromatic light are presented in Table 11.The fluorescein sample was illuminated with monochromatic light of anincident wavelength (490 nm) and the resulting emitted light was notmonochromatic or polarized and was composed of the wavelengthscharacteristic of fluorescein emission. Different sized spherical goldparticles were illuminated with monochromatic light of an incidentwavelength at which maximum scattering of incident light occurs and theresulting scattered light was either completely or partially polarized,depending on the size of the particle.

TABLE 10 MEASURED RELATIVE SIGNAL GENERATING POWER OF FLUORESCEIN VERSUSGOLD PARTICLES WHEN ILLUMINATED WITH WHITE LIGHT^((a)) ^((c))MEASUREDNUMBER OF FLUORESCEIN MOLECULES NECESSARY TO MATCH PARTICLE THE LIGHTSIGNAL DIAMETER INTENSITY FROM SOURCE (nm) ONE GOLD PARTICLE GOLD 10.1 ±<1.2 ˜9 GOLD 19.8 ± <2   ˜2.3 × 10² GOLD 29.5 ± <3.5 ˜4.1 × 10³ GOLD39.9 ± <6     ˜2 × 10⁴ GOLD 49.2 ± <9.8 ˜7.3 × 10⁴ GOLD  59.6 ± <11.9˜2.3 × 10⁵ GOLD  76.4 ± <15.3   ˜9 × 10⁵ PST^((b))   80 ± <6.6 ˜8.3 ×10³ ^((a))Incident light from an incandescent Leica microscope lightsource with a color temperature of 2,800° K., and the emitted light waspassed through a glass lens to decrease the infrared component.^((b))PST — polystyrene ^((c))Results not corrected.

TABLE 11 MEASUREMENT OF RELATIVE SIGNAL GENERATING POWER OF FLUORESCEINVERSUS GOLD PARTICLES WHEN ILLUMINATED WITH MONOCHROMATIC LIGHT^((c))MEASURED NUMBER OF FLUORESCEIN MOLECULES NECESSARY TO MATCH THELIGHT SIGNAL FROM PARTICLE ^((a))PARTICLE ^((b))INCIDENT ONE GOLD TYPEDIAMETER WAVELENGTH PARTICLE GOLD 10.1 nm ˜525 nm ˜4 GOLD 19.8 nm ˜530nm ˜120 GOLD 29.5 nm ˜532 nm ˜1,400 GOLD 39.9 nm ˜535 nm ˜7,800 GOLD49.2 nm ˜550 nm ˜25,000 GOLD 59.6 nm ˜542 nm ˜78,000 GOLD 76.4 nm ˜565nm ˜190,000 GOLD ˜100 nm  ˜560 nm ˜550,000 ^((a))Measurementsrepresented size ^((b))Incident monochromatic light composed of asignificant fraction of horizontally polarized light arising from themonochrometer. Vertically polarized incident light would yield asignificantly larger particle signal intensity but would not affect thesignal intensity from fluorescein. ^((c))Results not corrected.

Table 11 illustrates that the scattered light signal intensity generatedfrom various sized individual gold particles illuminated with incidentmonochromatic light is much more intense relative to the light signalintensity from a single fluorescein molecule. These results furtherillustrate that individual metal-like particles, as for example, goldparticles when illuminated with incident monochromatic light can bedetected to very low concentrations. Such detectabilty is extremelyuseful for using such particles with appropriate detection methods asextremely sensitive light scattering labels in diagnostic assays andanalytical applications.

Non-metal-like particles as for example, polystyrene which have 100's-1000's of highly fluorescent molecules incorporated into the body ofthe particle are well known in the art. An example of such a particle isa 110 nm diameter particle into which has been incorporated afluorescent compound which has excitation and emission wavelengthmaxima, 490 nm and 515 nm respectively, which are similar tofluorescein. Each particle contains an average of 4,400 highlyfluorescent molecules and the volume of such a particle is about 7×10⁻¹⁶cm³ and the fluorescent molecule concentration in the particle is about10⁻⁵ M. Table 12 presents the experimentally measured results for thelight generation power of 110 nm diameter polystyrene, polystyreneparticles loaded with many molecules of highly fluorescent compound, and100 nm diameter gold particles. The light generating power of these aredirectly compared against a solution of fluorescein which gives the samelight generation power. It is interesting to note that the totalscattered light signal from the 110 nm polystyrene particle alone isequivalent to the light signal from about 12,000 fluorescein molecules.The presence of the fluorescent molecules in the polystyrene particleonly increases the total light signal by about 1.5 fold of the particle.The fluorescence signal from this particle can be separated from thelight scattering signal by using the proper filter between the sampleand the detector which excludes incident light wavelengths and passesthe wavelengths characteristic of the fluorescence emission. Such afilter results in this particle generating a fluorescent signalintensity equivalent to about 3,000 fluorescein molecules. The 100 nmdiameter gold particle was clearly far superior in emitted lightgeneration power as compared to these particles.

TABLE 12 MEASURED RELATIVE SIGNAL GENERATING POWER OF FLUORESCEIN,POLYSTYRENE PARTICLES, POLYSTYRENE PARTICLES CONTAINING FLUORESCENTMOLECULES AND GOLD PARTICLES ^((c))MEASURED NUMBER OF FLUORESCENTFLUORESCEIN MOLECULES PARTICLE ^((a))PARTICLE MOLECULES INCIDENTNECESSARY TO MATCH THE LIGHT TYPE DIAMETER PER PARTICLE WAVELENGTHSIGNAL FROM ON PARTICLE ^((d))PST 110 nm 0 490 nm ˜12,000 PST 110 nm˜4400 490 nm ˜19,000 ^((e))Gold 100 nm 0 555 nm^((b)) ^((e))˜1.3 × 10⁶^((a))Measurements represented size ^((b))See Table 11(b) ^((c))Resultsnot corrected. ^((d))Polystyrene ^((e))The 100 nm diameter particle usedherein was from a different production batch than that used in Table 11.

Mixed Composition Particles

Spherical particles of mixed compositions were evaluated by theoreticaland physical experimentation to assess their possible utility in variousdiagnostic and analytic applications. For theoretical evaluations, agold “core” particle coated with different thickness of silver and asilver core particle coated with different thickness of either gold orpolystyrene were studied. By “core” is meant a spherical particle uponwhich an additional layer or thickness of different light scatteringmaterial is placed, resulting in a mixed composition of certainproportions. Direct physical experimentation was done for particlescomposed of a mixed composition where an additional thickness of silverwas added to a core gold particle of 16 nm diameter. In theseillustrative examples, gold and silver are representative of metal-likematerials and polystyrene is representative of non-metal-like materials.These examples are only a few of a larger number of different possiblecombinations which involve particles composed of mixtures of one or moredifferent metal-like and/or non-metal-like materials.

Results from calculations for the light scattering properties of theabove illustrative examples are presented in Tables 13 and 14. Table 13section A shows that for series of spherical 10 nm diameter particleswhich are composed of increasing proportions of a silver coat on a goldcore, the light scattering properties are changing to those more like apure gold particle. Most importantly, we observed in these calculationsand by physical experimentation, that certain proportions of a silvercoated gold particle can exhibit two intense light scattering maxima atincident wavelengths close to those characteristic of pure gold and puresilver particles of this rough size.

Direct experimental observation of 16 nm diameter gold particles coatedwith silver using white light illumination under DLASLPD conditions witha simple light microscope showed that there were new colors of scatteredlight from these particles which had not been previously seen in puregold or pure silver particle preparations. Many of the particles had abrilliant purple to magenta color of scattered light. Table 13 section Bpresents a comparison of the calculated results for mixed compositionparticles composed of a 10 nm diameter gold sphere coated with differentthickness of silver. The results show similar trends as seen in Table 13Section A for the light scattering properties of these mixedcompositions as the proportion of silver to gold is varied. Inadditional calculations (Table 14), where a silver core particle iscoated with varying gold thickness, the light scattering properties showsimilar trends in their changes as the proportion of gold and silver ischanged as seen in Table 13.

TABLE 13 CALCULATED SCATTERING PROPERTIES OF SPHERICAL PARTICLESCOMPOSED OF MIXED COMPOSITION — A GOLD CORE COATED WITH SILVER C_(sca)AT INCIDENT GOLD SILVER WAVELENGTH WAVELENGTH AT PARTICLE CORE COAT VOLGOLD MAXIMUM SCATTERING DIAMETER DIAMETER THICKNESS {overscore(TOTAL VOL)} (cm²) MAXIMUM(S) A 10 nm 10 nm 0 1 1.26 × 10⁻¹⁵ ˜530 nm 10nm  9 nm 0.5 0.73  7.3 × 10⁻¹⁶ ˜340 nm   8 × 10⁻¹⁶ ˜516 nm 10 nm 8.4 nm 0.8 nm 0.59   9 × 10⁻¹⁶ ˜340 nm  6.8 × 10⁻¹⁶ ˜510 nm 10 nm  4 nm   3 nm0.064  5.7 × 10⁻¹⁵ ˜380 nm B 10 nm 10 nm 0 1 1.26 × 10⁻¹⁵ ˜530 nm 11 nm10 nm 0.5 nm 0.75 1.25 × 10⁻¹⁵ ˜340 nm 1.45 × 10⁻¹⁵ ˜518 nm 12 nm 10 nm  1 nm 0.58  2.8 × 10⁻¹⁵ ˜340 nm   2 × 10⁻¹⁵ ˜505 nm 20 nm 10 nm   5 nm0.125  2.4 × 10⁻¹³ ˜375 nm Polystyrene Particle C 10 nm 0 0 —  1.3 ×10⁻¹⁷ ˜300 nm 20 nm 0 0 —  8.3 × 10⁻¹⁶ ˜300 nm

We have determined from our combined theoretical and physicalexperimentation the following. For particles composed of certain mixedcompositions of metal-like materials, as for example, mixed compositionsof gold and silver, new light scattering properties appear which areuseful in many different sample types and specific diagnostic andanalytic applications. Particles with two or more optically distinct andresolvable wavelengths of high scattering intensities can be made byvarying the composition of the metal-like-materials.

In contrast, particles composed of mixed compositions of non-metal-likeand metal-like materials generally exhibit light scattering propertiessimilar to the metal-like materials at equal proportions or less ofnon-metal-like materials to metal-like materials. Only at very highproportions of non-metal-like to metal-like materials do the lightscattering properties of the mixed composition particle resemble that ofthe non-metal-like material as the results of Table 14 section Bindicate.

Both the mixed silver-gold compositions and the silver-polystyrenecompositions exhibit the high light scattering power and visiblewavelength scattering bands which are characteristic of particlescomposed of pure metal-like materials. Particles of certain mixedcompositions are detectable by specifically detecting the scatteredlight from one or both of the scattering intensity peaks and or by thecolor or colors of these mixed composition type particles. Such mixedcomposition type particles enhances the capability for detecting lesseramounts of particles and more specifically, detecting lesser and greateramounts of particles than was previously possible.

Asymmetric Particles

The physical orientation of asymmetric particles with regard to a lightbeam allows for additional scattered light properties to be used in thedetection of these particles. The property of RIFSLIW can be used inmany different aspects of the current invention to more specifically andmore sensitively detect and or measure one or more analytes or particlesin a sample. For example, the flickering of the scattered lightintensity and/or change in color provides additional detection means todetermine which particles are bound to a surface and which particles arenot. This allows for non-separation type of assays (homogeneous) to bedeveloped. All that is required is to detect by particle counting,intensity measurements or the like the particles that do not flickerand/or change color. Unbound particles in solution will flicker and/orchange color while those bound to the surface will not. Additional imageprocessing means such as video recorders and the like allow foradditional methods of detection to be used with both asymmetric andspherical (symmetric particles). For example, In either a separation ornon-separation format, the bound particles are detected by focusing thecollecting lens at the surface and only recording those scattered lightsignals per unit area which are constant over some period of time.Particles free in solution undergoing brownian motion or other types ofmotion results in variable scattered light intensity per unit area perunit time for these particles. Bound light scattering particles arefixed in space and are not moving. By using image-processing methods toseparate the “moving” light-scattering particles from the “bound” lightscattering particles, the amount of bound particles is determined andcorrelated to the amount of analyte in the sample. One of skill in theart will recognize there are many other image processing methods thatcan be used to discriminate between bound particles to a surface andunbound spherical or asymmetric particles in solution.

Addition of Other Materials to the Surface or Core of the Particle toProvide Additional Physical Attributes not Related to the LightScattering Properties

In certain applications and with the use of certain types ofcompositions, it may be useful to “coat” the surface of a particle tofurther chemically stabilize the particle, or to add additional surfacebinding attributes which can be very important in specific applicationsto analytical diagnostic assays. For example, it is well known thatsilver rapidly oxidizes. For use of silver particles or particles ofmixed composition which contain silver, one can chemically stabilize thesilver-containing particle by applying a thin coat of gold or othersubstance on the surface such that the silver is no longer susceptibleto environmental effects on it's chemical stability.

In another example, one may want to coat the surface with anothermaterial such as a polymer containing specifically bound binding agents,or other materials useful for attaching binding agents, or the bindingagents themselves to the particles. In each of these examples, these“thin” coats do not significantly alter the light scattering propertiesof the core material. By “thin” coats is meant a monolayer or similartype of coating on the surface of the particle.

Manipulatable Light Scattering Particles (MLSP's) are particles which inaddition to having one or more desirable light scattering properties,these particles can also be manipulated in one-, two- orthree-dimensional space by application of an EMF. A MLSP particle can bemade in many different ways. For example, a MLSP particle is made bycoating a small diameter “core” ferroelectric, magnetic or similarmaterial with a much greater proportion of a material that has thedesirable light scattering properties, for example a 10 nm diameter coreof magnetic or ferroelectric material is coated with enough gold to makea 50, 70, or 100 nm diameter particle. This is shown in FIG. 29 A.

Another method of making such a particle is to coat the material thathas the desirable light scattering properties with a thin coat of themagnetic or ferroelectric material. For example, a gold or silverparticle of about 50 nm is coated with a 1-2 nm thick coat of themagnetic or ferroelectric material. This is shown in FIG. 29 B.

Alternatively, the MLSP particles are made by mixing in the appropriateproportions the light scattering desirable materials and theferroelectric or magnetic materials such that as the particle is formed,the appropriate proportions of light scattering desirable material tomagnetic or ferroelectric material per particle ratio is attained. Thisis shown in FIG. 29 C.

An alternative to the above MLSP particles is to link or assemble one ormore types of particles with desirable light scattering properties toone or more particles that can be moved by a EMF. Such multi-particlestructures can then have similar properties to the MLSP's. For example,small particles of magnetic or ferroelectric material are linked to oneor more particles who's light scattering properties are detected. Thelinking is by ionic, chemical or any other means that results in astable multi-particle structure. For example, the different particlesare coated with appropriate polymers so that when mixed in the properportion, a defined distribution of discreet multi-particle structuresare achieved by crosslinking the different types of individual particlestogether. There many different ways to link the particles together toachieve the desired multi-particle structure(s). For illustrativepurposes, a few of the possible multi-particle structures are shown inFIG. 30. FIGS. 30 A, B, and C show dimer, tetramer, and higher orderparticle constructs respectively for orientable MLSP particles. Oneskilled in the art will recognize that these are just a few of the manydifferent types of multi-particle structures possible and there arenumerous methods to make such structures.

These examples of particles composed of mixtures of one or more materialare but a few of a very large number of different compositions ofdifferent materials which are possible, and which would be apparent toone of skill in the art.

Particle Size and Shape Homogeneity

Depending on how the light scattering properties of particles aredetected, the approximate size and distribution of particle sizes in theparticle population can be extremely important. As an example, many ofthe commercially available gold particle preparations quote the particlesize distributions anywhere from about <10 to about <20 percentcoefficient of variation. Percent coefficient of variation is defined asthe standard deviation of the particle size distribution divided by themean of the particle preparation. Thus, for a 60 nm particle preparationwith a coefficient of variation of 20%, one standard deviation unit isabout ±12 nm. This means that about 10% of the particles are smallerthan 48 nm or greater than 72 nm. Such variation in size has significanteffects on the intensity of scattered light and the color of scatteredlight depending on the approximate “mean” size of the particles in thepreparation.

We have developed a particle growing procedure which seems to givenarrower size distributions than those available commercially. Theprocedure involves first making a preparation of “seed” gold particleswhich is then followed by taking the “seed” particle preparation and“growing” different size gold (see examples 11 and 15) or silverparticles (see Example 13) by chemical methods. For example, 16 nmdiameter gold particles are used as the “seed” particle and largerdiameter gold particles are made by adding the appropriate reagents (seeExample 15). This method is also very useful for making mixedcomposition particles.

Particle Homogeneity - Detection of Analytes by Scattered Light Color ofIndividual Particles

In certain applications, the color of the individual particles are usedto identify and quantitate specific types of analytes. For example, inimage cytometry applications, it may be of interest to identify andcount different types of cell surface antigens or the like by detectingthe number and color of different types of particles attached to thesurface. For this or any other related type of multi-analyte detection,the size distributions of the different particles need to be kept astight as possible. The average particle diameter of the particlepreparation should be chosen to provide the desired color of scatteredlight under white light illumination, using an average or “mean”particle size that is as close to the size midpoint between the meanparticle sizes of smaller and larger particles which will be used in thesame application to produce different colors of scattered light. In thisfashion, the resolvability of the different types of particles by theircolor of scattered light is maximized.

Particle Homogeneity-Integrated Light Intensity Measurement

In other sections we have described how the intensity of scattered lightcan vary greatly as particle size is increased or decreased. Thisvariation in the intensity must be taken into consideration especiallywhen integrated light intensity measurements are being performed. Usingthe 60 nm particle preparation described above with a 20% coefficient ofvariation, this means that 10% of the particles have intensities about 3times greater or less than a 60 nm particle. In addition, the particleswithin the remaining 90% of the population have quite varyingintensities. In applications where there are many particles beingmeasured, the “average” integrated light intensity should approximate a60 nm particle. However, at lower concentrations of particles, thestatistics of such a variation may affect the accuracy of the readingfrom sample to sample, and correction algorithms may be needed. By usingthe narrowest distribution of particles possible, the accuracy and easeof measurement is enhanced.

Useful Metal-like Particles for Detection of Analytes by their LightAbsorption Color

For some types of analyte assays, analytes are at concentrations wheredetection of the analytes by the light absorption properties can beaccomplished. For example, a current problem in the art ofimmunochromatographic assays and the like is that the use of goldparticles of the sizes typically used (4 to 50 nm diameter) onlyprovides for particles that can not be optically resolved by their lightabsorption color. These particles have a pink to red color when observedon filter paper or similar diagnostic assay solid-phase media. Byvarying the size and/or shape of silver particles and other metal-likeparticles many different colors of light absorption can be achieved.These different colors of the particles by light absorption can be usedto detect different analytes by the light absorption color of aparticle. These colors which can be detected by the eye are very usefulin many types of solid-phase assays such as immunochromatographic flowassays, panel type, and microarray or larger solid-phase single ormulti-analyte assays. Spherical and asymmetrical particles of silver andcertain mixed compositions of other metal-like particles allow for awide range of colors by light absorption.

Autometallographic Enhancement of Light Scattering Properties ofParticles

It is well known in the art that autometallography and relatedtechniques can be used to enlarge the size of existing metal-likeparticles by small or large factors. The light absorbing power ofparticles composed of metal and/or semiconductor material, and inparticular, gold and silver particles has often been used to quantitateand or detect the presence of these particles, by using either the eyeor an instrument designed to measure light absorbance. Such a method isinferior to the light scattering detection methods of the presentinvention in its ability to detect small numbers of particles enlargedby metallography.

As an example, it has been reported (see Immunogold-Silver Staining,Principles, Methods and Applications, CRC Press, 1995 M. A. Hayat Ed.)that one nanometer diameter gold particles were enlarged bymetallographic methods, coating the 1 nm diameter gold particles withsilver to an average diameter of about 110 nm in about twenty minutes.The particles in this preparation ranged in size from about 40 nm to 200nm diameter and were roughly spherical in shape. Surprisingly, ourcalculations show that enlarging the diameter of the 1 nm core tracerparticle to 110 nm results in an increase in scattering power of roughly10¹⁰ while the light absorption power is only increased by roughly 10⁵.

By increasing the diameter of a small particle, the incident wavelengthat which maximum light scattering occurs shifts to much longerwavelengths, as compared to a small core particle of the same material.Thus, enlarged particles are easily detected in the presence or absenceof small 1 nm particles by measuring the light scattering signal fromthe enlarged particles. The utilization for detection of the enlargedparticles of incident light of the wavelength at which maximumscattering occurs for the enlarged particles allows the more specificdetection of the enlarged particles relative to the smaller particleswhich constitute the major source of non-specific light scatteringbackground.

TABLE 14 CALCULATED SCATTERING PROPERTIES OF SPHERICAL MIXED COMPOSITIONPARTICLES — SILVER CORE PARTICLE COATED WITH GOLD OR POLYSTYRENE (PST)INCIDENT C_(sca) AT WAVELENGTH SILVER WAVELENGTH AT PARTICLE CORE COATCOAT VOL SILVER MAXIMA SCATTERING DIAMETER DIAMETER COMPOSITIONTHICKNESS TOTAL VOL (cm²) MAXIMA A 10 nm 10 nm — 0 — 1.1 × 10⁻¹⁴ ˜384 nm14 nm 10 nm Gold 2 nm 0.36 4.5 × 10⁻¹⁵ ˜372 nm   5 × 10⁻¹⁵ ˜518 nm 20 nm10 nm Gold 5 nm 0.125 6.5 × 10⁻¹⁵ ˜525 nm B 10 nm 10 nm — 0 1 1.1 ×10⁻¹⁴ ˜384 nm 10 nm  9 nm Gold 0.5 nm   0.73 1.9 × 10⁻¹⁵ ˜384 nm 10 nm 7 nm Gold 1.5 nm   0.34 5.6 × 10⁻¹⁶ ˜300 nm 6.8 × 10⁻¹⁶ ˜520 nm C^((a))10 nm 0 — 0 — 1.3 × 10⁻¹⁷ ˜300 nm PST 10 nm 10 nm — 0 0 1.1 ×10⁻¹⁴ ˜384 nm ^((a))20 nm 0 — 0 — 8.3 × 10⁻¹⁶ ˜300 nm PST 20 nm 20 nm —0 1   7 × 10⁻¹³ ˜382 nm 40 nm 20 nm PST 10 nm  0.125 9.3 × 10⁻¹³ ˜412 nm60 nm 20 nm PST 20 nm  0.037 1.25 × 10⁻¹²  ˜418 nm 20 nm 12 nm PST 4 nm0.216   4 × 10⁻¹⁴ ˜408 nm 20 nm 10 nm PST 5 nm 0.125 1.4 × 10⁻¹⁴ ˜410 nm20 nm  8 nm PST 6 nm 0.064 4.3 × 10⁻¹³ ˜414 nm ^((a))Particle composedof polystyrene only

Table 15 provides additional data with regard to the light scatteringproperties of small particles that are increased in size bymetallographic or related methods. Our calculated data show thatenlarging the size of a particle results in a greater increase in theparticle's light scattering power as compared to it's light absorptionpower. For example, a twenty percent increase in particle diameterincreases the small particle light scattering power by (1.2)⁶ or aboutthree-fold. An increase of two and ten-fold in small particle diameterwill result in a scattering power increase of about sixty-four and onemillion-fold respectively, while the light absorbing power is increasedby only eight-fold and one thousand-fold respectively.

Thus, when the method of the present invention is used to quantitate andor detect the presence of particles which have been enlarged bymetallography (that is, the deposition of a coat of a metal-likematerial onto a small diameter composed of either metal-like ornon-metal-like materials), it is possible to detect lesser amounts ofsuch particles, and to more specifically detect lesser amounts of suchparticles, than was previously possible.

The above is an illustration of the combination of metallographicenlargement of a metal-like particle core by the deposition of ametal-like coat on the core, followed by the detection of the enlargedparticles by the method of the present invention. Such methods can beused for enlarging particles free in solution and or particles attachedto a surface. The preceding example is only one of the many differentpermutations of this combination method which include the use of themany different strategies and methods discussed herein for detectingparticles by light scattering as well as different combinations of coreand coat compositions and different degrees of enlargement. Thesealternate combinations will be readily apparent to one of skill in theart. Such a combination approach can be applied to almost any situationwhere it is desirable to use a signal generation and detection system todetect an analyte.

TABLE 15 2 nm DIAMETER GOLD TRACER CORE PARTICLE — DIFFERENT THICKNESSOF A SILVER COAT OF UNIFORM THICKNESS — CALCULATED LIGHT SCATTERINGPROPERTIES PARTICLE WAVELENGTH PARTICLES RELATIVE LIGHT AT WHICHRELATIVE DECADIC MOLAR DIAMETER THICKNESS SCATTERING SCATTERINGSCATTERED EXTINCTION OF OF SILVER POWER (C_(sca)) MAXIMUM LIGHTCOEFFICIENT OF PARTICLE COAT (cm²) OCCURS INTENSITY PARTICLE  2 nm  0 nm  ˜8 × 10⁻²⁰ ˜520 nm 1 1 10 nm  4 nm ˜10⁻¹⁴ ˜382 nm ˜1.25 × 10⁵ ˜3 ×10^(2(a)) 20 nm  9 nm ˜6.5 × 10⁻¹³ ˜384 nm  ˜8.1 × 10⁶ ˜2.3 × 10³ 40 nm19 nm ˜2.8 × 10⁻¹¹ ˜400 nm  ˜3.5 × 10⁸ ˜1.6 × 10⁴ 80 nm 39 nm ˜2.9 ×10⁻¹⁰ ˜447 nm  ˜3.6 × 10⁹ ˜5.8 × 10⁴ 100 nm  49 nm ˜4.3 × 10⁻¹⁰ ˜481 nm ˜5.4 × 10⁹ ˜7.6 × 10⁴ 150 nm  74 nm ˜7.9 × 10⁻¹⁰ ˜432 nm  ˜9.9 × 10⁹˜1.5 × 10⁵ 150 nm  74 nm ˜7.6 × 10⁻¹⁰ ˜600 nm  ˜9.5 × 10⁹ ˜1.2 × 10⁵^((a))The molar decadic extinction coefficient, the C_(sca) and theincident wavelength at which maximum light scattering occurs areessentially identical to those for a pure 10 nm diameter silverparticle.

Method of Refractive Index Enhancement

The use of refractive index matching techniques in light microscopy,telecommunications, and other related fields is well known in the art.This technique is generally used to decrease the nonspecific lightscattering and reflections that occur as a light beam passes from onemedium or device to the other as for example, from the surface of onematerial to the surface of another different material.

We have determined that the light scattering power (C_(sca)) of aspecific type of particle is affected by the medium in which theparticle resides. Altering the refractive index of the medium results ina change in a particle's light scattering properties.

Table 16 provides an illustrative example of medium refractive indexeffects on selected particles. Calculated refractive index mediumeffects for gold, silver, and polystyrene spherical particles of 10 nmdiameter are presented.

The effects of the refractive index of the medium are quite differentfor metal-like particles as compared to non-metal-like particles asTable 16 shows. Increasing the refractive index of the medium formetal-like particles as for example gold, results in increasing theintensity and wavelength maximum of the light scattered from theparticle while for a non-metal-like particle, as for examplepolystyrene, the light scattering power is decreased.

The unique light scattering properties of metal-like particles ascompared to non-metal-like particles as an effect of the refractiveindex of the sample medium can be used to more specifically and withgreater sensitivity detect metal-like particles in samples includingthose which have high non-specific light scattering backgrounds. This isimportant for many different types of diagnostic analytical assays.

TABLE 16 CALCULATED MEDIUM REFRACTIVE INDEX EFFECTS FOR TEN NANOMETERDIAMETER PARTICLES OF DIFFERENT COMPOSITION. WAVELENGTH AND INTENSITYEFFECTS GOLD SILVER POLYSTYRENE N₁ (A) (B) (A) (B) (A) (B) 1 1 520 nm 1355 nm 1 400 nm 1.1 1.9 525 nm 1.6 360 nm 0.9 400 nm 1.2 3.9 525 nm 2.3370 nm 0.75 400 nm 1.3 7.7 530 nm 2.9 380 nm 0.52 400 nm 1.4 15.1 535 nm3.9 390 nm 0.27 400 nm 1.5 27.7 540 nm 5.3 400 nm 0.084 400 nm 1.6 45.4550 nm 7.3 415 nm ˜0 — 1.7 71.5 555 nm 9.7 425 nm 0.1 400 nm (A) =Relative scattering power at different medium refractive indices (B) =Wavelength at which scattering maximum occurs N₁ = refractive index ofmedium

In many types of samples and diagnostic assay formats, the problem ofnon-specific scattered, reflected, and other background light fromsample containers and non-analyte sample constituents are well known.These non-specific light backgrounds make it difficult, if notimpossible to perform sensitive to ultrasensitive detection of analytesby detection and/or measurement of the scattered light properties of aparticle.

We have determined that metal-like particles can be detected to muchgreater specificity and sensitivity as compared to non-metal-likeparticles when the method of refractive index enhancement is used. Themethod is now described. The effect of the refractive index of theparticle and medium on the scattered light intensity can be evaluated byusing the following expression (RI is refractive index factor)$\begin{matrix}{{RI} = {{refmed}^{4}{\frac{m^{2} - 1}{m^{2} + 2}}^{2}}} & (16)\end{matrix}$

where refmed is the refractive index of the medium, and m is equal tothe refractive index of the particle/refmed. m depends implicitly onwavelength but the exact dependence varies with particle composition andmedium. The refractive index of most solvents which have no color isusually independent of wavelength, at least in the visible region of thespectrum.

It is of interest for the use of light scattering particles in sensitiveassays to determine which values of refractive index leads to higherlight scattering intensities. This is determined from the refractiveindex factor (RI) of Eq.(16). This factor has it's highest value whenthe denominator of Eq.(16) is zero. For this condition, the refractiveindex factor has an infinite value. Thus, the condition for high lightscattering is

m ²+2=0   (17)

Solving above equation for m, we get $\begin{matrix}{m = \sqrt{- 2}} & (18) \\{\text{~~~} = {1.41}} & (19)\end{matrix}$

where i =−1. The above equation indicates that the refractive indexfactor has its highest value and light scattering from the particle isat a maximum when the refractive index is a pure imaginary number with avalue of 1.41. The calculated data presented in Table 16 do follow theexpected trends. In addition, the method of refractive index enhancementworks very well at incident wavelengths far removed from the incidentwavelength at which maximum light scattering occurs for the metal-likeparticles.

An illustrative example of the use of the refractive index enhancementmethod is now provided. In highly scattering samples, such as sampleswhere there is a high level of non-specific light scattering background,metal-like particles and the method of refractive index enhancement areused as follows.

One skilled in the art increases the refractive index of the samplemedium as for example, placing a film of water or other liquid on top ofa dry or wet sample. This increases the refractive index of the medium.In another example, a serum or other type of highly scattering sample isdiluted with a high refractive index liquid which substantiallyincreases the refractive index of the medium.

For the above mentioned examples, the following processes occur. Thespecific light scattering signal of the metal-like particles increasesand the non-specific light scattering background decreases as therefractive index of the sample is increased. The largest increases inparticle light scattering/non-specific scatter background ratio isachieved when the refractive index of the sample medium approaches therefractive index of the metal-like article as demonstrated in Table 16.This means that at the proper medium refractive index values, thenon-specific light scatter from serum proteins or similar constituentscan be significantly reduced or eliminated while the specific lightscattering intensity of the particles is increased. This results insuperior analyte detection signal/background ratios when the lightscattering properties of metal-like particles are used as the analyticaltracer. These methods can be applied to samples such as dry surfaces,surfaces covered by solutions, or solutions.

These index matching methods can also be used with longer wavelengthsfor the metal-like particles to increase the specific light scattersignal/non-specific scatter background ratio even further. While Table16 only shows the effects for gold and silver particles, particlescomposed of other metal-like materials can also be used to detect lesseramounts of particles using the methods we have described. Thedescription of the method of refractive index enhancement describedherein presents only a few of many possible variations of this practiceof the invention. Many other variations of the method are possible andwill be apparent to one of skill in the art. One or another of thesevariations can be effectively utilized in most diagnostic formats todetermine the presence or absence of an analyte. This aspect of thepresent invention provides a means for the detection of lesser amountsof particles, and for the more specific detection of lesser amounts ofparticles, and for the more specific detection of lesser and greateramounts of particles than was previously possible.

A method of the present invention which combines refractive indexenhancement with the narrow band pass filter approach described earlierhas great utility for detection of lesser and greater amounts ofparticles than was previously possible. These approaches arecomplimentary. The refractive index enhancement method is used todecrease non-specific light scattering background while the narrowfilter is used to reduce and minimize other sources of non-specificlight background such as fluorescence and the like. A combination ofthese methods results in highly optimized particle specific scatteringsignal/non-specific light background ratios and allows for the morespecific and more sensitive detection of particles.

The above example is only one of many possible variations of thiscombined method. Other variations will be apparent to one of skill inthe art.

Detection of Light Scattering Particles in Highly Scattering andFluorescent Samples - Serum

Mammalian serum contains many medically important substances whosequantitation and or presence is determined in the clinical laboratory aswell as elsewhere. Many different signal generation and detectionsystems are used to determine the presence of these analytes in serumand these include light signal generation methods such as fluorescence,light scattering, and chemiluminescence, as well as calorimetric methodswhich are used in formats involving both direct labeling and signalamplification methods. Natural serum contains a variety of substanceswhich are capable of producing a non-specific light signal by eitherfluorescent, chemiluminescent or light scattering mechanisms. Inaddition, the serum often contains substances which interfere with thegeneration and or detection of the specific light signal from the tracerentity. These difficulties make it difficult if not impossible toconduct analyte detection in pure or nearly pure serum samples.

In order to be able to effectively employ most, if not all, of theexisting test systems for detection of serum analytes, it is almostalways necessary to pre-process the serum in some way to make itsuitable for testing. Many such serum processing methods exist andperhaps the simplest is dilution of the serum into some appropriatesolution which is usually aqueous in nature. Another commonly usedapproach is to conduct the actual test in such a manner that theundesirable serum components are removed before the presence of thespecific light producing tracer is determined. From a cost and laborpoint of view, the less effort and reagents needed to conduct the test,the better. It is highly desirable not to pre-process the sample at all.Such capability could also be beneficial to the performance of the test.The method of the present invention provides a means to conduct suchanalyte tests in almost pure serum and further provides a means, todetect lesser or greater amounts of particles more specifically in highconcentrations of serum than was previously possible.

For example, it is common to dilute serum samples to a finalconcentration of about 5 percent serum before analyzing it with afluorescent tracer such as fluorescein. The serum sample is illuminatedwith monochromatic light at 490 nm, and optical filters are used tominimize non-specific scattered light background. The non-specific lightsignal is equivalent to a highly pure liquid sample of fluorescein whichcontains 10⁻⁸ M to 10⁻⁹ M fluorescein. Thus, in the 5% serum sample, onecan detect 10⁻⁸ M to 10⁻⁹ M fluorescein at a signal to noise ratio of 2.In a 95 percent serum sample, the lower limit of detection offluorescein would be about 19 times higher, or about 1.9×10⁻⁷ M to1.9×10⁻⁸ M. Thus, in 95 percent serum with optical filters, the lowerlimit of detection of fluorescein is about 1.9×10⁻⁷ M to 1.9×10⁻⁸ M andthis amount of fluorescein light signal results in a (total lightsignal) to (non-specific light signal) ratio of about 2 to 1.

Table 17 presents the experimentally measurable detection limits, at a(total light signal) to (non-specific light signal) ratio of 2 to 1, offluorescein at very high serum concentration. At this high serumconcentration, and in the absence of optical filtration to removenon-specific light signal due to scattered incident light, the lowerlimit of detection of fluorescein is about 6×10⁻⁷ M.

Table 18 presents the lower limit of detection of fluorescein at veryhigh serum concentration when an optical filter which eliminates thenon-specific light signal due to scattering of incident light is placedbetween the sample and the photomultiplier tube. In this situation, thelower limit of detection of fluorescein in high serum concentration isabout 2×10⁻⁸ M.

In contrast to fluorescein, the results presented in Table 17 Section Band Table 18 demonstrate that in the absence of optical filtration thepresence of 59.6 nm diameter gold particles in 95 percent serum can bedetected with a (total light signal) to (non-specific light signal)ratio of 2 to 1 at a concentration of about 1.8×10⁻¹² M. Thenon-specific light signal observed from the serum was equivalent to thatfrom about 5×10⁻⁷ M fluorescein. Under these same conditions 60 nmdiameter polystyrene particles in high serum concentration can only bedetected at a lower limit of about 6×10⁻⁹ M (see Table 18).

TABLE 17 DETECTION OF 59.6 nm DIAMETER GOLD PARTICLES AT HIGH SERUMCONCENTRATION PERCENT GOLD PARTICLE INCIDENT FLUORESCEIN RELATIVE LIGHTSERUM CONCENTRATION WAVELENGTH CONCENTRATION INTENSITY A 97.8% 0 490 nm0 1.02 95.7% 0 490 nm 8.7 × 10⁻⁶M 15.8 97.8% 0 545 nm 0 0.52 95.7% 0 545nm 8.7 × 10⁻⁶M 0.56 B 97.8% 0 490 nm 0 0^((c)) 95.8% 1.77 × 10⁻¹²M 490nm 0 1.1 97.8% 0 543 nm 0 0.55 95.8% 1.77 × 10⁻¹²M 543 nm 0 1.05 Fetalbovine serum purchased from Biowhitaker, Walkerville, MD catalog number14-501F. Serum was passed through a one micron filter before sale andwas clear but straw colored. Serum pH was adjusted to about ph 9 to 9.5.No wavelength filtration of the emitted light was done ^((a))The lightsignal obtained here represents a value of 1. This signal was equivalentto 3.7 × 10⁻⁷M fluorescein.

TABLE 18 LOWER LIMIT OF DETECTION OF FLUORESCEIN, GOLD AND POLYSTYRENEPARTICLES AT 92.8% SERUM CONCENTRATION TYPE AND FILTER PERCENTFLUORESCEIN CONCENTRATION OF INCIDENT FOR RELATIVE^((c)) SERUMCONCENTRATION PARTICLES WAVELENGTH EMISSION SIGNAL INTENSITY 92.8% 0 0554 nm NO 1 (˜520 mυ)^((b)) 92.8% 0 Gold 1.8 × 10⁻¹²M 554 nm NO 2.1(˜1100 mυ) 92.8% 0 0 496 nm YES^((a)) 1 (˜39 mυ) 92.8% 2.3 × 10⁻⁸M 0 496nm YES ˜2 (˜79 mυ) 92.8% 0 0 554 nm NO 1 (˜468 mυ) 92.8% 0 PST 6 × 10⁻⁹M554 nm NO ˜2 (˜960 mυ) Polystyrene (PST) and gold particles had ameasured diameter of 60 nm and 59.6 nm respectively. The pH of thesolutions containing fluorescein was adjusted to pH 9-10 beforemeasurement. The maximum light intensity in serum was observed at anincident wavelength of about 496 nm for fluorescein and about 554 nm forthe 59.6 nm gold particle. ^((a))The light signal emitted from thesample was passed through a No. 16 Wratten filter before encounteringthe photomultiplier tube. ^((b))The instrument measurements were allobtained at identical instrument settings and are directly comparable toone another (mv = millivolts). ^((c))The light detection instrumentdetects fluorescein emissions slightly more efficiently than theparticle emitted light. In addition the incident monochromatic light isenriched for horizontally polarized light and this reduces the particleresults due to a lower level of light scattering from the particles butdoes not affect the fluorescein light intensity. The total instrumentbias towards the fluorescein signal is roughly 1.5-2 fold.

Results presented in Table 19 present a comparison of the relativedetection limits (at total light signal to non-specific light signalratio of about 2 to 1) of 100 nm diameter gold particles, 110nm diameterpolystyrene particles, and 110 nm diameter polystyrene particlescontaining 4,400 molecules of highly fluorescent compound per particle,in 95.7 percent serum. These results further demonstrate that the 100 nmdiameter gold particles can be detected at a much lower concentrationthan 110 nm diameter particles composed of polystyrene or polystyrenecontaining many molecules of highly fluorescent compound. The goldparticles can be detected in serum at about 230 times lowerconcentration than the other non-metal-like particles.

Table 20 compares the amount of scattered light measured from identicalconcentrations of 59.6 nm gold particles in a solution containing a highconcentration of serum and a solution containing only water under thesame illumination conditions. Under these conditions, a gold particleconcentration of 1.8×10⁻¹² M was detectable at a signal/background ratioof about 3. These results indicate that the presence of serum or any ofthe common constituents does not appear to have any direct effect on thelight scattering power of the gold particles. Such stability andinertness of the light scattering properties of metal-like particlesmake them extremely useful in samples such as serum and other relatedsamples which contain many other constituents.

The detection of 100 nm diameter spherical polystyrene or gold particlesin serum provides a further illustrative example.

Mammalian serum contains around 3.7 gram percent of protein, of whichabout two-thirds is serum albumin. The detection of polystyreneparticles in serum is hampered by the non-specific light scatteringwhich originates from protein and other substances in serum, as well asmany other sources. The similarity of the light scattering intensityversus the incident visible wavelength profiles for polystyreneparticles and the proteins and other substances in serum severely limitsthe ability to detect the polystyrene particles in serum or any otherhighly scattering medium.

TABLE 19 DETECTION OF PARTICLES COMPOSED OF GOLD, POLYSTYRENE (PST), ANDPOLYSTYRENE CONTAINING A FLOURESCENT COMPOUND AT HIGH SERUMCONCENTRATION PARTICLE RELATIVE PERCENT DIAMETER AND PARTICLE INCIDENTLIGHT SERUM COMPOSITION MOLARITY WAVELENGTH INTENSITY^((d)) 100%   0 0490 nm 1^((e)) 580 nm 0.27 95.7% 110 nm PST^((b)) 1.9 × 10⁻¹¹M 490 nm1.9  580 nm 0.54 95.7%    110 nm PST^((a)) + 1.9 × 10⁻¹¹M 490 nm 2.2 fluor 580 nm 0.54 95.7% 100 nm gold^((c)) 8.2 × 10⁻¹⁴M 496 nm 1.1  580nm 0.59 ^((a))Obtained from Interfacial Dynamics Corp., Portland,Oregon. Each particle contains an average about 4400 fluorescentmolecules. The excitation and emission maxima are 490 nm and 515 Nnmrespectively for the fluorescent molecules. The fluorescent compoundconcentration in the particle is about 3 × 10⁻²M. ^((b))Obtained fromInterfacial Dynamics Corp. ^((c))Produced by art known methods. ^((d))Nowavelength filtration of emitted light was done. ^((e))The light signalobserved here represent a value of one. All other values are relative tothis value. This signal is equivalent to that from about 2 × 10⁻⁷Mfluorescein.

TABLE 20 SIGNAL GENERATION FROM 59.6 nm DIAMETER GOLD PARTICLES AT HIGHSERUM CONCENTRATION AND AT ZERO SERUM CONCENTRATION PERCENT GOLDPARTICLE INCIDENT RELATIVE TOTAL SERUM CONCENTRATION WAVELENGTH SIGNALSIZE 0     1.8 × 10⁻¹²M 543 nm 1 95.7% 0 543 nm 0.58 95.7% 1.8 × 10⁻¹²M543 nm 1.38 Gold particles had a measured diameter of 59.6 nmrespectively. 95.7% serum is straw colored and has an optical density at1 cm pathlength and wavelength of 543 nm of about 0.14, The limitscattering measurements were made in a 6 mm by 50 mm glass tube with aninner diameter of about 5 mm. It is estimated that absorbance of lightby the serum reduces the scattered light signal by about 15 percent.

The use of an incident wavelength of 575 nm instead of 300 nm toilluminate the serum results in an about 13 fold reduction in thenon-specific light scattering signal, but also results in about the sameextent of reduction for the specific scattering signal from thepolystyrene particles. Increasing the wavelength of illumination forpolystyrene or other non-metal-like particles does not appear tosignificantly increase the specific signal to background ratio, that is,the detectability of the polystyrene particles in the sample.

In contrast, metal-like particles are detected to greater signal tobackground ratios as compared to non-metal-like particles by increasingthe visible wavelength of illumination and/or detection. A 100 nmdiameter gold particle maximally scatters light around wavelengths ofabout 575 nm in aqueous media similar to water. Illumination of thesample with monochromatic light of wavelengths around 575 nm results inthe generation of the maximum light scattering signal from the goldparticles and significantly reduces the non-specific light scatteringsignal. For example, under these conditions, the total non-specificlight scattering is reduced by about thirteen-fold as compared to anillumination wavelength of 300 nm relative to an incident wavelength of300 nm.

The illumination of the serum sample with incident white light andappropriate optical filters which minimize the amount of light outsideof the wavelengths of interest (less than and or greater than aspecified band centered at about 575 nm) provides another means todetect lesser amounts of metal-like particles in serum. Under theseconditions the incident visible wavelength which produces the maximumlight scattering intensity from the gold particle is utilized, and thenon-specific light scattering signal originating from serum protein andother substances as well as other sources is greatly reduced. Multipletypes of different metal-like particles are detectable in serum sampleswhen illuminated by white light (or several different wavelengths) andusing an appropriate array of optical filters. This method makes use ofeach type of particle having a different incident wavelength at whichmaximum light scattering occurs.

Another approach involves filtering the total light signal from thesample through a proper polarization filter and/or a bandpass filter.Use of the proper polarization filter will result in the effectiveremoval of unpolarized fluorescence background but will have littleeffect on the non-specific light-scattering background since it ismostly polarized. When using broad band illumination, as for example,white light illumination, using optical band pass filters of higherwavelength allows for significant reduction of the non-specific lightscattering and fluorescence background. Many of the metal-like particleshave high light scattering intensities at longer wavelengths and thisproperty can be utilized in combination with the bandpass filter and/orpolarization filter approach. For example, a spherical gold particle of300 nm diameter has near maximum scattering efficiency at a wavelengthof about 700 nm and it's scattered light intensity is about six timesthan a 100 nm diameter gold particle. Using the 300 nm particle and abandpass filter centered at 700 nm decreases the non-specific light byhalf and increases the gold particle scattering power by a factor of 6(as compared to the 100 nm particle). Thus, the signal to backgroundratio in this system has been increased by a factor of 12. Use of thisapproach with non-metal particles, for example, polystyrene ofcomparable size, does not significantly increase the signal tobackground ratio but may actually lower it. The use of anti-reflectivecoating on the optical components of the apparatus, and/or samplechamber may also improve the signal to background ratio. Many otherschemes and approaches are also possible and these would be apparent toone of skill in the art.

This aspect of the invention results in improved discrimination betweenthe specific light scattering signal and the non-specific lightscattering background signal of a diagnostic assay system over thatattainable by other methods which use the detection of scattered lightas part of a test system format. In addition, the availability ofdifferent types of metal-like particles which exhibit different colorswhen illuminated by white light makes it possible to detect the presenceof multiple types of particles in one sample, which has utility fordetecting multiple analyte types in one sample.

A further advantage of particles of metal-like particles is the chemicalinertness of these particles, relative to fluorescent compounds. Suchparticles do not photobleach and their signal generation capacity is notaffected by such mechanisms.

The above approaches are just a few examples of the many possibleapproaches for using particles composed of metal-like materials toimprove discrimination between the specific light scattering signal dueto the particle, and the non-specific light scattering signal which canoriginate from a variety of sources. For example, a large number ofschemes are possible in which such particles are specifically detectedat a wavelength different from the wavelength at which maximum lightscattering occurs for the particle being used. Many other schemes orapproaches are also possible and these would be apparent to one of skillin the art.

The detection of one or more analytes in a solid-phase or related sampleby detection of one or more of a light scattering particle's propertiesis now discussed.

Solid-Phase Detection Methods

In the previous sections we have described various aspects of theinvention as they relate to certain light scattering properties ofmetal-like particles, and the detection of these particles in asolution. We now describe our methods for detection of particles thatare on a surface or very close to a surface.

We have determined that by using gold, silver, and other metal-likeparticles with our methods of DLASLPD illumination and detection, we areable to detect very low quantities of particles and particle-labeledbinding agents (coated particles) per unit area, being able to detectsingle particles and particles coated with binding agents on or near asurface using simple illumination and detection means. These methods canbe used on either optically transmissive or non-optically transmissivesurfaces.

We have determined that with the use of certain combinations ofparticles and methods of illumination and detection, we can detect awide range of particle densities from about 0.001 to 10³ particles persquare micron (μ²) in a sample. By using the proper type(s) ofparticles, different types of analytes can be detected to very lowlevels and across very wide concentration ranges in the same sample, asfor example, in microarrays. This is accomplished on one apparatus byutilizing both particle counting (at low particle densities) andintegrated light intensity measurements (at high densities) on the samesample. For example, if a sample is to be analyzed for two or moredifferent analytes by using solid-phase related means such as arraychips or other solid-phase methods, different types of analytes exist atdifferent concentrations in the samples. That is, some analytes may beat higher or lower concentrations from a couple to a few orders ofmagnitude as compared to other analytes in the sample. The selection ofthe proper types of particles is extremely important in achieving thedesired analyte detection sensitivity and range of concentrations themethod will work for. Our methods as we describe herein provides fordetection of analytes in such samples. Even wider detection ranges andgreater sensitivities are possible if more powerful light sources suchas lasers are used, and more sophisticated detection methods such asconfocal imaging are added to our basic illumination and detectionmethods.

We have determined that we can detect high densities of particles morespecifically and easily, that is, with very good signal to backgroundratios using simple methods. In some aspects of the present invention, acollection lens(imaging lens, mirror or similar device) is used and inother aspects, a collection lens is not used.

The scattered light from the particles is detected by a photodetector asfor example, a photodiode or photodiode array, photomultiplier tube,camera, video camera or other CCD device, or the human eye. The amountof particles is determined by counting the number of particles per unitarea and/or measuring the total integrated light intensity per unitarea. The specific scattered light properties detected and measured areone or more of the following: the scattered light intensity at one ormore wavelengths, the color, the polarization, the RIFSLIW, and/or theangular dependence of the particle scattered light per unit area. Thisis then correlated to the presence, absence, and/or amount of theanalyte(s) in the sample.

In some assays where one or more analytes is to be determined, one orboth of the particle counting or integrated light intensity measurementscan be used. It should be noted that with proper selection of particlesand the use of DLASLPD illumination and detection methods, there isusually so much optically resolvable and detectable scattered lightintensity available that more sophisticated light sources, and spatialand optical filtering techniques are not necessary. However, in somesamples where there may be significant amounts of non-specific lightbackground, the ultimate signal to background is improved by usingoptical filters, confocal imaging, or other aperture type spatialfiltering techniques to increase the particle scattered light signal(s)to total non-specific light background ratio.

In some analytical and diagnostic applications, the scattered lightintensity can be detected and measured using our basic methods withoutthe use of a collection lens or mirror. In these samples, one or moreproperties of the scattered light is detected and measured in the samemanner as described above without the use of a collection lens. Themethods are now discussed in more detail.

Detection of Scattered Light from Light Scattering Particles using aCollection Lens or Mirror

We have found that we can use various types of light collection opticaldevices to collect the scattered light of the particles. We have usedboth particle counting and intensity measurements (integrated intensityper unit surface area) to detect one or more of the specific lightscattering properties of the particles in a given area with our methodsof DLASLPD illumination and detection. We have found that in most of theexperiments we have done, that it is generally useful to use thecounting measurement method when the particle densities are about 0.1particles per μ2 or less. When the particle densities are greater thanabout 0.1 particles per μ2, we find that measuring the total integratedlight intensity is a useful measurement method. It should be notedhowever, that one can use the counting measurement or integrated lightintensity measurement methods at particle densities greater than or lessthan about 0.1 particles per μ².

The use of a specific type or types of lens to collect and/or image thescattered light from the sample we have found useful relates to thefield or area of the surface we are interested in measuring, the type ofsample container that is being measured, and the upper limit of particledensities that are to be measured by particle counting. For example, ifwe are interested in measuring larger areas to detect the scatteredlight, a ×10 or even smaller microscope objective or lens or mirror canbe used to collect the scattered light from the sample. Similarly, if asmaller areas of the sample is to be measured, a ×20, ×40, ×100, orgreater microscope objective lens or similar lens or mirror can be usedto collect the light. If the method of particle counting is to be usedat higher particle densities, greater power objective lenses allow forbetter resolution of the particles at high densities. It should be notedthat when larger objectives are used, additional requirements andlimitations come into play. For example, the working distance becomesvery small and immersion oil may be needed to be added to the sample.When a camera, video camera, or similar CCD type photodetector is used,the total scattered light from the sample area is detected. Thisinformation can then be processed by simple hardware and/or softwaremeans to analyze the scattered light measurements. This is a powerfulcapability, because many different analytes in a sample can be detectedand quantitated by use of a solid-phase microarray, array chip, orsimilar format. In the microarray format, small areas of the surface areeach covered by a different type of binding agent in a spatiallydistinct region that specifically binds a particular analyte. Wedescribe later specific applications of the present invention tosolid-phase multi-analyte microarrays and the like.

The method of particle counting is usually more instrumentally demandingthan the method of integrated light intensity measurement. However, forvery sensitive detection of one or more of the light scatteringproperties of a particle, there are many advantages to using thecounting technique. For example, fluctuations and inhomogeneities in thelight source or sample chamber do not effect the particle countingmeasurement whereas these problems can cause severe problems when themethod of integrated light intensity measurement is used. In addition,there are many software and hardware options to enhance the quality andsignal/background ratios of the measured particles by countingtechniques.

Detection of Light Scattering Particles without the Use of a CollectionLens or Mirror

We have also developed methods where the use of a collection lens is notnecessary to detect the scattered light of the particles at or near asurface. In this arrangement we usually detect the scattered lightcoming from the area of interest by the integrated light intensity. Thiscan be done by the naked eye, or a photodetector as previouslydescribed. We have found that when we use metal-like particles that areabout 120 nm in diameter or less, we can significantly increase theparticle light scattering signal to total non-specific light backgroundratio by placing our detector (either eye or photodetector) at anglesoutside of the envelope of the forward direction of the scattered light.

Key Concepts for Increasing Signal/Background Ratios

Before we describe the DLASLPD methods of illumination and detection indetail, it is useful to summarize the key concepts that when used in oneform or another determine the signal and signal to background ratiolimits for the detection of the light scattering particles. Thesemethods are in addition to adjusting or changing various apparatuscomponents such as using a more powerful light source, a more highlycollimated light source, a smaller wavelength band light source, adifferent wavelength light source, a more sensitive photodetector,optical and/or spatial filters between the illumination source and thesample and/or between the sample and the detector, and/or confocal orsimilar imaging techniques. These strategies and methods are outlinedbelow.

(1) by the use of larger diameter metal-like particles, the lightscattering power of the particle can be significantly increased. Theincrease in size may also change the scattered light intensity versusincident wavelength profile. These properties can be adjusted to suitthe need of any particular assay such that one or more of the scatteredlight properties are easily detectable. For example, for the measurementof analytes in samples with high non-specific light backgrounds, alarger gold particle, about 80-120 nm or greater in diameter is useful.The maximum wavelength at which maximum light scattering occurs shiftsto higher wavelengths and the intensity of the scattered light alsoincreases as compared to a 40 nm diameter gold particle. The combinationof these two effects significantly increases the signal/background ratioas compared to the 40 nm diameter gold particle.

(2) by measuring the scattered light of the sample at angles outside theenvelope of the forward direction of the scattered light, thesignal/background ratio in either the intensity or counting mode issubstantially increased. We have observed that the detector can beplaced either above or below the surface plane of the sample as well ason the same side or opposite side of the sample plane where theillumination beam is located. In these various orientations, thespecific light scattering signals from the particles are detectedoutside the envelope of the forward direction of the scattered light,while most of the non-specific scattered light from optical aberrationsin the sample chamber and other constituents in the sample are withinthis envelope of the forward direction of scattered light. This allowsfor more sensitive and specific detection of the particle scatteredlight.

(3) the amount of non-specific reflected light also affects thesensitivity of detection as we have previously described. We have foundthat the amount of reflected light can be substantially reduced bymoving the incident light surface as far as possible away from thecollection area that is being detected. This can be accomplished in manydifferent ways, including the proper design of the sample chamber(discussed later). For example, we noticed that if we put a thin layerof immersion oil on the bottom of a glass slide, through which the lightbeam illuminates the particles on the opposite surface, we saw highlyimproved results. In another experiment, when we glued a small plasticlight guide to the bottom of a plastic sample chamber with a microarrayof bound particles on the opposite side of the surface, we saw veryimproved results. We have also used much larger optical alignment meanssuch as an equilateral prism and/or other types of prisms or opticallight guides and placed immersion oil at the surface where the samplecontainer interfaces with the prism. We have concluded that thesesuperior results are a result of (i) having the light incident surfaceremoved a greater distance away from the area of detection that containsthe light scattering particles; (ii) having an angle of incidence of 0degrees on the surface of the light guide such as a prism face and thelike (with respect to the perpendicular) and (iii) that much of thereflected light which occurs in the system is guided out of the systemand away from the collection point. All of these methods are useful inincreasing the signal/background ratios for the detection of lightscattering particles in various samples. There are several light guidingstrategies that can be used to effectively remove the reflected lightout of the system to improve signal to background.

(4) refractive index enhancement methods are also extremely useful inincreasing the signal to background ratios in many different types ofsamples. We have found several methods to increase the signal tobackground, a few of which are now discussed. The use of liquid to coverthe surface containing the particles, where the closer the liquid'srefractive index is to the refractive index of the surface that containsthe particles, the better the signal/background ratio. We have foundthat for detecting analytes on a dry solid-phase, the signal/backgroundratio is improved by placing a liquid layer on top of the surface. Forexample, when an aqueous buffer solution of refractive index of about1.33 is used to cover the sample surface we get much improved results ascompared to measuring the particles on the same surface in air. Evenbetter signal/background ratios are obtained by using liquids which moreclosely match the refractive index of the solid-phase. For example, anassay can be performed by first binding the analytes with lightscattering particles coated with binding agent to the solid-phase in thesample medium or other appropriate reaction mixture or buffer. Thesolution in the sample is then diluted or replaced with a solution ofchosen refractive index that covers the solid-phase prior to detectionof the particles. In this fashion, highly sensitive results can beachieved.

In addition to the above methods, the further use of narrow band passoptical filters, cutoff optical filters, spatial filtering such asapertures either between the illumination beam and the sample and/orbetween the sample and the photodetector or eye will also increase thesignal/background ratio. Use of confocal imaging techniques may also beuseful in certain analytical assay applications where the cost andsophistication of such techniques and apparatus are not an issue. Theuse of longer wavelength sources either optically filtered or not arealso ways to increase the signal/background ratio. Guiding the excessnon-specific light out of the system by using specifically designedsample chambers to remove the excess light is another useful method.General sample chamber designs that are useful are described later. Allof these variations on one or more aspects of the current inventionprovide for increased signal/background ratios and thus provides for themore specific and sensitive detection of one or more analytes in asample.

There are many different ways the DLASLPD illumination and detectionmethods can be specifically applied to a sample and these are outlinedin FIG. 15. FIG. 14 provides a diagram for orientation and descriptionof the outline of the DLASLPD methods shown in FIG. 15. One skilled inthe art will recognize the utility the method affords when used withcertain metal-like particles to detect one or more analytes in asolid-phase or similar sample.

The detail of the methods is now described.

Illuminating and Light Collection Optics

1. General Concepts

The solid-phase methods we now describe can be applied to the detectionof light scattering particles. The detection and measurement of one ormore light scattering properties is then correlated to the presence,absence, or concentration of one or more analytes in a sample. Thesemethods can be used with most, if not all known solid-phase analyticmethods including microarray, array chip, or similar formats. The methodis designed to have a wide range of sensitivities (from low sensitivityto the ultra sensitive range). This range of sensitivities is achievedwith easy to use and inexpensive apparatus.

In the technology, the number or relative number of particles on asurface is determined through methods that depend on the lightscattering properties of particles. The detection system consistsfundamentally of (1) a magnifying lens (also called an imaging orcollection lens) that forms a magnified image of the light scatteringparticle patch or a portion of the patch and (2) an illuminating systemthat makes the particles appear as bright objects on a dark background(the DLASLPD method). The method can be performed without the need forthe collection lens also. The number of particles in the magnified imagecan be quantified by particle counting or by measuring the scatteredlight intensity (which is proportional to particle number or density).Particle counting can be done by (a) eye (unaided or with an ocularlens, depending on particle size), (b) an electronic imaging system (forexample video camera, CCD camera, image intensifier) or (c) aphotosensitive detector with a field limiting aperture and a scanninglight beam arrangement. Scattered light intensity can be measured withan electronic imaging system or photosensitive detector. At low particlesurface densities (less than about 0.1 particles per μ²), the particlecounting method is preferred while at higher surface densities(especially, where the individual particles are closer than the spatialresolution capabilities of the magnifying lens) the steady lightscattering intensity measurement is preferred. The technology isdesigned to easily shift between these two methods of detection, thatis, between particle counting and intensity measurements and can be usedwith particle diameters down to about 20 nm depending on the lightscattering power of the particles and specific hardware components ofthe detection apparatus.

Light Illumination Systems

The illuminating system is a key element in the technology. Theilluminating systems are designed to illuminate a particle patch orgroup of particle dots with high light intensity in such a manner thatthe individual particles appear as bright objects on a dark background.This allows visualization of particles attached to a surface or free ina fluid film above the surface. Free particles are distinguished fromattached particles by their Brownian motion which is absent in attachedparticles. In the following sections we describe the details and logicof the illuminating systems.

Applicant has experimented with many different illumination systemsincluding an expensive commercial dark field illuminator called anultracondenser (Zeiss) . Two fundamental methods of illumination andseveral versions of these two methods can be used. These methods aresimpler and seem to produce higher illuminating light intensities than,for example, the ultracondenser.

General Description of the Fundamental Illuminating Methods

The illuminating systems are designed to (1) deliver a beam of highlight intensity to a patch (or group of dots) of light scatteringparticles and (2) minimize the amount of the illuminating light thatenters the detecting system directly or through reflections. This isachieved by constraining the light beam and its reflections to anglesthat are outside the light collecting angles of the detecting system. Inone illuminating method, the collecting lens and the light source are onopposite sides of the solid-phase surface (illumination from below) andin the other method, the illuminating light source and magnifying lensare on the same side of the surface.

Direct Illumination from Below the Magnifying Lens

FIG. 1 presents a schematic diagram of one of the basic methods ofillumination used. In this method, the light impinges on the solid-phasesurface S from below the surface. It is assumed that S is transparent(although it could have some color). O is a region on the surface thatcontains light scattering particles. The magnifying or light collectinglens L is located above S. The angles at which L collects light areshown as a shaded cone C (light collecting cone of lens L) with an apexat the surface S (where the light scattering particles are located) anda base determined by the diameter D of the lens. The illuminating lightbeam (LB) is angled so that is does not enter the light collecting coneof L. The arrows show the direction of travel of LB.

The solid-phase can be, for example, a microscope slide, a microtiterplate or other types of transparent solid-phases used in clinicaldiagnostics. The light source can be any type of light source, such asfilament lamps, discharge lamps, LEDs, lasers and the like. Light iscollected from the illumination light beam using optical light fibersand light collecting lenses, and then focused onto the scattering lightparticles using a condenser lens. The mean angle θ which the light beammakes with the surface S is adjusted so that the beam of light does notenter the lens L as explained above. The adjustment of the angle θ caneasily be done by visual observation of the light scattering particlesthrough the magnifying lens and occular (compound microscopearrangement),adjusting the angle so that the particles appear as brightobjects on a dark background. This angle also serves well for lightscattering intensity measurements although at high particle densities,the focusing requirement is not as stringent.

The magnitude of the angle θ can be deduced from the numerical apertureof the magnifying lens. For ultrasensitive detection, a microscopeobjective is used as the magnifying or imaging lens. A microscopeobjective usually has its numerical aperture inscribed on its housing.Numerical aperture can be defined in terms of the diagram of FIG. 2.This figure shows a magnifying lens (with focal length f) that isfocused on a patch of light scattering particles at O. The distancebetween the lens and O is equal to f. The lens (L) collects all lightscattered from O into the solid cone whose base is the diameter D of thelens. The angle θ_(H) is defined as the planar half angle of this solidcone. The numerical aperture (N.A.) of the cone is related to θ_(H) bythe expression

N.A.=n sin(θ_(H))   (36)

where n is the refractive index of the medium between the lens and thepoint O. The medium, for example, can be air (n=1), water (n=1.33) orimmersion oil (n=1.5). For small values of θ_(H), N.A. is approximatelyequal to D/2f where D is the diameter of the lens and f is focal length.

The following table gives typical values for the numerical apertures andθ_(H) of objectives that can be commonly used (for n=1).

Magnification N.A. θ_(H) in degrees ×10 0.25 14.47 ×20 0.5 30 ×40 0.6540.54 ×63 0.85 58.2

As already mentioned, the exciting light beam must be angled so that itis outside of the light collecting solid cone of the magnifying lens.For high magnifications, the angle at which the exciting beam impingeson the solid-phase surface must be large. For example, for a ×40objective, the incident angle must be larger than 40°. Since thefraction of light reflected at a surface increases with incident angle,we must consider whether the angles that have to be used in ourilluminating system result in a large loss of light due to reflection.Furthermore, we must consider whether critical reflections (totalinternal reflections) are involved at high incidence angles. Thefollowing is a brief discussion of the fundamental laws of refractionand reflection needed in the subsequent discussion of the effects ofreflections in this illuminating system.

Snells Law of Refraction

We describe Snells law of refraction in terms of the diagram of FIG. 3.This figure shows a light beam that travels along a medium of refractiveindex ni (i for incident medium) and impinges on the surface S of amedium of refractive index nt (t for transmission medium). Part of theincident light is transmitted into medium t (the refracted beam) andpart is reflected (the reflected beam) back into medium i. If the angleof incidence is θi then the angle of the refracted beam is given bySnells Law which can be written as

ni Sin(θi)=nt Sin(θt)   (37)

If ni<nt then θi<θt. If ni>nt then θi>θt. Note that angles are measuredwith respect to a line that is perpendicular to the surface S. Thereflected beam makes an angle θr=θi (that is, the reflected and incidentangles are equal).

Laws of Reflection. Fraction of Incident Light that is Reflected at aSurface.

The fraction R of incident light intensity which is reflected fordifferent incident angles θi can be calculated using Fresnels equationsof reflection. (It should be noted that intensity is here defined asenergy per unit time per unit area. Intensity is also calledirradiance). However for simplification, we present our discussion interms of plots relating R to θi. The exact dependence of R on θi isdetermined by the values of ni and nt and the state of polarization ofthe incident light. Important facts concerning reflectance are asfollows.

i. Reflectance for the Case where the Light Beam Travels from a Mediumof Low Refractive Index to One of High Refractive Index (ni<nt).

FIG. 4 shows plots of R vs. θi (ω=θi) for the case where ni=1 (air) andnt=1.5 (the latter is close to the refractive index of glass or plastic)and for light polarized parallel (rp) and perpendicular (rs) to theplane of incidence. The plane of incidence is defined as the plane whichcontains the incident light beam and the line perpendicular to thesurface (see FIG. 3). The reflectance R of unpolarized light is given bythe average of the graphs for light polarized parallel and perpendicularto the plane of incidence. In FIG. 4, the reflectance graph forunpolarized light is labeled Ord (for ordinary). The graphs of FIG. 4,show that

a. rs increases continuously with increasing ω(ωin FIG. 3 is the same asθi as used herein). The increases in rs is small up to about 70° (wherethe reflectance is only about 15%) and then increases much more rapidlyreaching 100% reflectance at 90°. Thus, the fraction of light that isreflected is less than 20% up to incidence angles of 60°.

b. rp decreases with increasing ωup to about 57° where rp is zero. Theangle at which rp=0 is called the Brewster angle or polarizing angle.The Brewster angle θb can be calculated with the expression

Tan(θb)=nt   (38)

assuming that ni=1 (air). For nt=1.5, above equation gives θb=56.3. Itshould be noted that at the Brewster angle, θi+θt=90°. Thus for nt=1.5,θi=θb=56.3° and θt=33.7°. For angles greater than the Brewster angle, rpincreases rapidly with increase in ω and reaches a value of 100% at 90°.

c. For unpolarized (ordinary light), the reflectance increased graduallywith increase in ω up to about 70° and then increases rapidly reaching100% at 90°. Less than 20% of the incident light is reflected forθi<70°.

d. It should be noted that the intensities of the reflected andtransmitted light do not add up to the intensity of the incident light.This seems to violate the law of conservation of energy. This apparentviolation is actually due to the definition of intensity as energy perunit time per unit area. Because of refraction, the incident andtransmitted light do not have the same cross sectional area. If thedifferences in cross sectional areas are taken into consideration, thenit can be shown that the energy per unit time in the reflected andtransmitted beams add up to the energy per unit time in the incidentbeam.

ii. Reflectance for the case where the light beam travels from a mediumof high refractive index to one of low refractive index (ni>nt).

FIG. 5 shows plots of reflectance of polarized light vs. angle ofincidence (ω=θi) for ni=1.54 and nt=1. The plots are quite differentthan those of FIG. 4 for ni<nt. The most significant difference is thatat an incident greater than about 41° all of the light is reflected(100% reflection, total reflection). The smallest incident angle atwhich total internal reflection occurs is called the critical reflectionangle θc. The value of this angle depends on the values of ni and nt. Anexpression for calculating θc from values of ni and nt can be derived byconsidering the angles of the incident and transmitted light beams atthe critical angle. At the critical angle θc, the reflected beamcontains most of the incident light and makes an angle θc with therespect to a line perpendicular to the surface as required by the lawsfor specular reflection. The transmitted light has low intensity and itsangle θt with respect to perpendicular line is 90°. That is, thetransmitted light beam travels parallel to the surface. We can thereforeobtain the value of θc by inserting θt=90° in Snell's equation. Thisinsertion gives

nt Sin(90)=ni Sin(θc)   (39)

Since Sin(90)=1, we can write Sin(θc)=ni/nt   (40)

For nt=1.54 and ni=1 (air), the above equation gives θc=40.5°. It shouldbe noted that the critical angle is the same for unpolarized light andlight polarized perpendicular or parallel to the incident plane. Thatis, θc is independent of whether the light is unpolarized or planepolarized.

iii. Effects of Reflectance and Refraction on the Illumination of aPatch of Light Scattering Particles.

We first consider the simple case where the particles are on the surfaceof a dry microscope slide in air. That is, the particles are dry and airis the medium on both sides of the microscope slide. FIG. 6 shows aschematic diagram of the reflections and refractions involved in thiscase. The first reflection occurs at the surface S1 (ni<nt, ni=1 andnt=1.5). FIG. 4 shows that the fraction of light reflected is below 20%for incident angles up to 70°. Therefore, reflections at S1 are notproblematic in this method of illumination. Surface 2 could beproblematic because the light beam passes from a low to a highrefractive index and the possibility exists for total internalreflection at this surface. The critical angle for total internalreflection at a surface where ni=1.5 and nt=1 (air) is about 42°(calculated with Eq. (40)). The question now is whether this criticalangle is attained when the incident light beam at surface 1 has a largeangle.

FIG. 7 shows a plot of θi2 [θtj] vs. θi1 [θij] calculated with SnellsEq.( (37)) and using θt1=θi2. As can be seen from the plot, θi2 rapidlyincreases with increase in θi1 up to about θi1=70°. The increase in θi2then levels off and does not reach the critical angle until θi1=90°.However at θi1=90°, no light is transmitted across S1. We thus concludethat for the arrangement of FIG. 4, critical illumination is neverachieved at any practical angle of θi1. Furthermore, reflections do notsignificantly diminish the amount of light delivered to the particles onS2 for θi1 values less than about 70°.

We have verified the above conclusions experimentally. In ourexperiments, however, we have found that light scattered by smudges,dirt, scratches and other irregularities or artifacts on surfaces S1 andS2 (non-specific light scattering) can become comparable to the lightscattered by the particles on S2 and thus significantly increases thebackground light and diminishes the sensitivity of particle detection.However, the non-specific light scattered by artifacts on S1 and S2 isconcentrated in the forward direction of the illuminating light beamwhereas the light scattered by the particles (for small particles) is inall directions. These effects are demonstrated in FIG. 8.

In FIG. 8, the intensity of non-specific light scattered by surfaceartifacts in any direction θ is given by the length of the line (withangle θ) extending from the origin O to the intensity envelope. Thelight scattered by the particles is shown as dashed lines which go outin all directions from O. The effects of non-specific light scatteringon the detection of specific light scattering can be demonstratedexperimentally using a microscope slide containing 60 nm gold particleson the surface. In air, these particles scatter green light, and in theabsence of non-specific light scattering, an illuminated patch ofparticles appears as a green patch on a dark background. Surfaceartifacts scatter white light and when this type of non-specific lightscattering is superimposed on the specific particle light scattering,the light scattered from a patch of particles has a greenish-white colorinstead of a pure green color. The effects of preferential forwardscattering by surface artifacts can be seen by viewing the scatteredlight by eye positioned at different angles θv as shown in FIG. 8. Whenthe eye is placed at θv=0, the scattered light has a greenish whitecolor. As the angle of observation θO is increased, the white colordecreases and, at θO greater than 30°, only the pure green color of goldparticles is seen. Thus, in the present invention, it is useful toobserve by eye or detect with photodetector means at an angle greaterthan θv of 30°. As we show later, non-specific light scattering due tosurface artifacts can be further reduced by illuminating through lightguides as for example, a prism type of arrangement.

We next consider the case where the light scattering particles are in athin film of water that is on a microscope slide and covered with acover glass as shown in FIG. 9. The illuminating beam encounters foursurfaces where changes in refractive index occur, namely S1 (air toglass), S2 (glass to water), S3 (water to glass), S4 (glass to air).Consideration of the reflection and refraction at each surface as donein previous paragraphs leads to the conclusion that in the system ofFIG. 9, reflections do not significantly reduce the amount of lightenergy delivered to the scattering light particles and that criticalreflection does not occur at any surface. Non-specific light scatteringdue to surface artifacts on surfaces S1 and S4 are present as in thecase of FIG. 8. However, the presence of water greatly reducesnon-specific light scattering for surfaces S2 and S3.

Direct Illumination from the Same Side as the Magnifying Lens

This method of illumination is shown in FIG. 10. The meaning of S, L, C,and LB are the same as in FIG. 2. In this Figure, the exciting lightbeam impinges on the surface S from above the surface. The lightcollecting lens is also above S. The exciting light beam is angled sothat neither the incident or reflected light enter the light collectingcone C of the lens L. In this method of illumination, it is necessary tokeep the light reflected from the different surfaces in the path of thebeam from being reflected into the light collecting cone C of themagnifying or imaging lines L. Since the angle of reflection is the sameas the angle of incidence at each surface, collection of unwantedreflected light by L can be minimized by confining the illuminatinglight beam to those angles which are outside of the cone C. It can beshown, as in the previous section, that reflections do not significantlyreduce the amount of energy delivered to the light scattering particlesand that critical reflections do not occur in dry or water coveredparticles. Nonspecific light scattering due to surface artifacts are thesame as discussed in the previous section.

Illumination through a Prism Arrangement.

i. Illumination from below.

FIG. 11 presents a schematic diagram of a prism setup. In one of itssimplest forms, it consists of a triangular prism on which can sitmicrotiter wells, glass slides, microarrays on plastic or glasssubstrates and the like which contain the light scattering particles tobe detected. The sample chamber or slide that contains the lightscattering particles is located on the upper surface S2 of the prism,the surface containing the particles is at the focal point of the lensL. Immersion oil is placed between the sample chamber or slide and theprism to minimize non-specific light by refractive index matching. Theparticles are dry in air. If the index matching at S2 is exact or almostexact, then the light beam does not undergo significant refraction orreflection at S2. Thus, an illuminating light beam travels in anapproximately straight line as it transverses the prism and surfacecontaining the light scattering particles. However, there is refractionat the air-prism interface S1 and at S3. Consider an illuminating lightbeam which enters the prism in a direction which is perpendicular to S1(angle of incidence is 0°) and suppose that the prism is a 45° prism(angle γ=45°). Because the beam travels in a straight line from S1 tothe point O on S3, it strikes the surface S1 at an angle of 45°. Thebeam will then undergo total internal reflection since the criticalangle for glass to air is about 42°. Thus, in contrast to illuminationfrom below without a prism (see FIG. 6), the prism arrangement allowscritical reflection. Recall, that in the absence of a prism (FIG. 6),critical reflection cannot be achieved because of the refraction oflight at S1 as shown in FIG. 6 (also see FIG. 7). In order to deliver ahigh energy light beam when using a light guide such as a prism, theilluminating light beam must be directed so that it strikes S3 (FIG. 11)at an angle less than 42°. The question now is whether incident anglesless than 42° at S3 allow one to satisfy the condition that the lightwhich exits at S3 must be outside the collecting cone of the lens L.Suppose that the angle of incidence at S3 is 35°. From Snells law (withni=1.5 and nt=1) we calculate that the exit angle is 62° which isoutside the collecting cone of our most demanding objective, namely thex40 objective with θH=41°. We conclude that the prism arrangement ofFIG. 11 permits delivery of high light energy to dry light scatteringparticles in air while maintaining a dark background.

We now consider the prism arrangement of FIG. 11 in which the lightscattering particles are covered with water and a cover glass. Asdescribed before, an exciting light beam travels in a straight line fromS1 to O where it encounters the glass-water interface. It then travelsthrough the water and cover glass, finally encountering the glass-airinterface at the upper surface of the cover glass. It is of interest toconsider the reflections that occur at the glass-water and glass-airinterfaces. The angle for critical reflection at the air water interfaceis equal to 62.5° (using ni=1.5 and nt=1.33 in Eq.(40)). Theintroduction of water at the surface S3 thus permits illumination atmuch higher angles, than in air, without encountering total internalreflection. Furthermore, at angles less than 62.5°, reflectance is lowat the glass-water interface. We now consider the reflection at thecover glass-air interface. Consider a beam of light which entersperpendicular to the surface at S1. If the prism is a 45° prism, thenthe beam strikes S3 at an angle of 45° where it undergoes total internalreflection. Refraction at S3 (glass to water) changes the angle of thebeam to 55°. However, refraction at the water-cover glass interfacebends the beam back to 45°. The beam thus strikes the cover glass-airinterface at 45°. The prism arrangement with particles in water and acover glass thus permits efficient delivery of light energy to the lightscattering particles (attached to the surface S3 or free in water) butthe incident light is totally reflected at the cover glass.

From the above discussion we conclude that there are no reflections thatseriously affect the delivery of light energy to light scatteringparticles in a film of water. Total reflection does occur at the coverglass-air interface but these reflections do not affect the delivery ofincident light energy to the scattering particles.

From the above discussions, it is evident that both prism and non-prismarrangements permit efficient delivery of energy by angled illuminationof particles attached to a surface or free in solution. By efficientdelivery we mean that the beam of light does not undergo total internalreflection at any pertinent surface and the collection of non-specificlight is minimized. However, we have found experimentally that superiordetection capabilities occur with the prism arrangement in certainapplications because it eliminates or considerably reduces artifacts dueto scattering from irregularities on glass or plastic surfaces close tothe light scattering particles. The immersion oil used to couple thesolid-phase to the prism, eliminates almost completely non-specificlight scattering from irregularities at S2 (FIG. 11). These effects seemto be due to an index matching mechanism. The non specific lightscattering at the point of entry of the light beam at S1 is far removedfrom the specific scattering particles at point O (if the prism issufficiently large) and does not contribute to the scattered lightcollected by the magnifying lens. Furthermore, if the specificscattering particles are in water, index matching by the water filmconsiderably reduces non-specific scattering on the surface S3.Moreover, in the presence of water and a cover glass, the illuminatinglight beam undergoes total reflection at the cover glass-air interface.This reflection considerably diminishes non-specific scattering fromirregularities at the cover glass-air interface because only a smallamount of light energy reaches the irregularities on this surface. Inaddition, the total internal reflection diminishes or eliminates theprobability that illuminating light can enter directly the lightcollecting cone of the magnifying lens. It should be noted that totalinternal reflection can also affect the collection angle of the lens Lbecause particle scattered light which makes an angle greater than 42°at the cover glass-air interface is totally reflected. This effecthowever is not a serious one.

Microscopic Observations

In the previous section we discussed our illumination and detection(magnifying lens) systems with emphasis on the factors (reflections andrefractions) that govern the efficient delivery of light energy to lightscattering particles (which are attached to a surface or free just abovethe surface) while minimizing the non-specific light that is collected.In this section we present experimental details obtained by visualobservation, through an ocular, of the magnified image produced by themagnifying lens L.

a. Details of Light Sources and Optical Fibers

We have used three different types of light sources as follows.

i. Leica Microscope Illuminator

This is a standard, commercial microscope illuminator. It uses atungsten filament light bulb and lenses that produce a 5×7 mm focusedimage of the filament at a distance of about 22 mm from the tip of theilluminator. To produce a more focused light beam, we attached a x10objective lens to the microscope. The lens is about 6.5 cm from the tipof the illuminator. The objective produces a focused spot of light ofabout 4 to 5 mm in diameter at a distance of about 7 mm from theobjective lens.

ii. Bausch and Lomb Fiber Lite Illuminator

This is also a commercial illuminator. It uses a 150 watt tungstenfilament lamp that is mounted on a parabolic reflector. The reflectorproduces a beam of almost parallel light with a diameter of about 25 mm.An 11 mm diameter optical light guide (consisting of many small opticalfibers bundled together) is positioned close to the filament lamp. Theoptical light guide is then split into two equal light guides, each witha diameter of about 5.3 mm and a length of about 2 feet. We use one ofthe light guides. To produce a focused light spot, we collimate thelight from the optical fiber using a 25 mm focal length lens (20 mmdiameter) positioned about 25 mm from the end of the optical fiber. Thecollimated light is then focused, into a 5 mm diameter light spot, by ax10 objective which is positioned about 50 mm from the collimating lens.The collimating and objective lenses are in a compact holder that isrigidly attached to the optical fiber. The flexibility of the opticalfiber makes this type of light source system much easier to use than therigid Leica Microscope Illuminator.

iii. Custom Illuminator

This is an illuminator which we constructed. It uses a 12 V, 28 Watttungsten filament lamp that is mounted on a parabolic reflector. Thereflector produces an almost parallel beam of light which is focusedinto an optical guide that has a diameter of 0.125 inches. The opticalguide is 36 inches long. Light from the reflector is focused unto theoptical fiber with a lens (23 mm focal length, 9 mm diameter) that ispositioned close to the lamp. The light guide has a numerical apertureof 0.55 and accepts a cone of light with a planar half angle of 60°.Light which exits at the other end of the light guide is collimated by a12 mm focal length lens (diameter 11 mm) positioned about 17 mm from theend of the fiber. The collimated light is finally focused into a brightspot of 5 mm diameter by a x 10 objective. The objective lens ispositioned at a distance of about 26 mm from the collimating lens. The 5mm bright spot is at a distance of about 12 mm from the end of thefocusing lens.

b. Prisms (light guides)

FIG. 12 shows diagrams of some light guides such as prisms which we havefound useful in our illuminating systems. Some of these are not actuallyprisms in the classical sense but may rather be called light guiders orlight guides. The light guider allows light to be delivered efficientlyat an angle while allowing the reflected light beam to exit at the faceopposite to the incident light face. It must have some minimumdimensions for the following reasons. The spots where the light beamenters and reflected light exits can produce significant non-specificlight scattering due to surface irregularities. These spots must besufficiently removed from the patch of specific light scatteringparticles to minimize their non-specific light scattering contribution.The light guides can be molded into one piece with the sample chamberthus eliminating the need to use immersion oil between the light guiderand analysis piece. We have proto-typed such a device by gluing a smalllight guide to the bottom of a plastic chamber which has a microarray ofstreptavidin spots coated on the inner surface of the sample chamberwell. Our detection and measurement of bound light scattering particlesto the individual microspots of the microarray using this device wereessentially the same as our measurements by particle counting andintensity measurements with the sample chamber placed on a prism withimmersion oil between the two surfaces.

c. Microscopic Observations

Using an ocular for visual microscopic observations, we have evaluatedseveral illuminating arrangements with special emphasis on brightness ofparticles, darkness of background, and usefulness in different types ofclinical assay formats. We have found several arrangements that givegood results. Here we will limit our discussion to one of the easiest touse and least expensive arrangements which gives excellent results. Byexcellent results we mean bright particles on a dark background usingx10 and x40 objectives as magnifying lenses.

The imaging system is an inexpensive microscope from Edmund Scientific.The microscope consists of an objective (x10 or x40) and an ocular in astandard 160 mm tube. The reason for using a microscope, instead of justsimply an objective and an ocular, is the convenience provided by thefine/course focusing mechanism of the microscope. However, we havemodified the stage of the microscope to adapt it to our method ofillumination and have replaced the microscope condenser with a lightguide (prism type) as shown in FIG. 12(d). The cylinder below the prismfits into the condenser holder. The modified stage and illuminator makeit possible to work with microscope slides, microtiter plates and otherplastic plates. However, the x40 objective cannot be used with the thickplastic plates because of the small working distance (about 0.45 mm) ofthis objective. x40 objectives with longer working distance areavailable. The custom Illuminator is used for illumination.

To setup the microscope system for the DLASLPD method, a microscopeslide containing free or surface bound gold particles (60 nm goldparticles in a thin water film covered with a cover glass) is placed onthe microscope stage. The prism is positioned so that its surface isalmost in contact with the microscope slide. The slide and prismsurfaces are coupled with immersion oil. The x10 focusing objective ofthe illuminating system is positioned so that it is almost in contactwith the side of the prism that is illuminated (see FIG. 13). Theobjective is angled so that the light enters perpendicular to theilluminating surface and strikes the surface S (surface in contact withthe microscope slide) at an angle of about 45°. If the gold particlefilm on the slide has a sufficiently high concentration of particles(about 6×10⁹ particles/ml or higher) the spot where the light crossesthe particle film displays a strong yellow-green color due to lightscattering by the particles. The position of the x10 focusing objectiveis adjusted so that the yellow-green spot is centered with respect tothe microscope (magnifying) objective. The microscope is then focused onthe spot so that the particles appear as sharp objects when viewedthrough the ocular. The position and angle of the x10 illuminatingobjective is than repositioned to produce bright objects on a darkbackground. This adjustment is repeated with the x40 objective of themicroscope. There is a narrow range of positions which produce brightobjects on a dark background for both x10 and x40 objectives.

It should be noted that none of the illuminating light is transmittedinto the air space above the microscope slide when the illuminatinglight beam strikes the prism surface at greater than about 42° (criticalangle for total internal reflection at the plastic or glass-airinterface). In our arrangement, the angle of incidence is 45°. This canbe verified by placing a piece of white paper above the microscopeslide. No illuminating light falls on the paper. However, it is ofinterest to visualize the illuminating beam to determine its shape orprofile in space. This can be done by placing a rhodamine plastic blockon top of the slide using immersion oil for coupling. The rhodamineblock is a transparent plastic block that contains the fluorescentmolecule rhodamine. A beam traveling through the block can be seen fromthe rhodamine fluorescence that it produces. The immersion oileliminates the air space and allows the illuminating beam to enter theplastic block. The profile of the illuminating beam seen by fluorescenceinside of the block is shown in FIG. 13.

Once the illuminating system has been properly positioned, goldparticles greater than about 30 nm can easily be seen on microscopeslides, plastic wells, and solid-phase microarrays or array chips. Thex10 objective permits detection of particles densities less that 0.005particles per μ². The x10 objective has a working distance of about 8.5mm so that it can be used with plastic pieces that are about 8 mm thick.

Method of DLASLPD Video Contrast Enhancement

We have determined that metal-like particles and non-metal-likeparticles can be detected to greater sensitivities in samples by usingthe method of DLASLPD Video Contrast Enhancement. This method involvesadjusting the imaged non-specific light background electronically suchthat it is essentially removed from the imaged field while keepingindividual particles visible. The method works extremely well with theother methods as described herein. We have observed improved specificityand sensitivity results using this method.

Sample Chamber Improvements

In the spirit of taking certain aspects of the present invention and nowfurther improving it in terms of ease of use, adaptability to differenttesting environments and conditions, we have found that several aspectsof the present invention can be embodied in the design of samplechambers that are used to conduct the assay and detect the analytes.

For example, based on our observations and conceptualizations we canapply our principles to the general design of the sample chamber, thatis the container which contains the sample to be analyzed. Theseimprovements can facilitate the ease of use and applicability to thetypes of tests and testing conditions briefly outlined above. It shouldbe stated however, that the invention as described herein can bepracticed equally well without use of the following sample chamberimprovements. These improvements are for the means of increasing thepractical applicability of the present invention to specific testingconditions and environments and described elsewhere herein.

We have found that by moving or displacing the surface S1 (incidentlight surface) as far as possible away from the area that contains theparticles to be measured, the signal/background ratio is significantlyincreased. We have previously described the use of an optical alignmentmeans such as a prism or similar optical light guide which is used toassist in orienting the delivery of the illumination beam to the surfaceS1. Usually one will use immersion oil between a surface of the lightguide (prism etc.) and a surface of the sample chamber. There arenumerous conditions in analyte testing where it may be preferable to nothave immersion oil as a component in the analytical methodology. We havedetermined that by increasing the thickness of the surface upon whichthe particles are on or nearby significantly reduces the level ofnon-specific light as described earlier.

We have applied this and other aspects to the design of sample chambersfor the detection of one or more analytes in a sample. These generalsample chamber designs are diagrammed in FIGS. 17, 18, and 19. FIG. 17shows a sample chamber that has beveled flat sides. The degree of angleof the beveled sides is matched to the angle of illumination such thatthe illumination beam strikes the face of the beveled side at an angleas close to 0 degrees as possible (with respect to the perpendicular).In this fashion non-specific reflected and scattered light is minimized.FIG. 18 shows a sample chamber that has curved sides instead of the flatbeveled sides as described for FIG. 17. In this sample chamber, for theexit beam which is diverging, the curved surface allows for the moreefficient removal of this non-specific light. FIG. 19 shows a samplechamber that utilizes both concepts of moving the incident surface wherethe light beam strikes the sample further away from the area to bemeasured and the curved sides to allow for more efficient removal ofnon-specific light. Thus, this sample chamber has an increased thicknessof material below the bottom surface of the well, beveled flat surfacesbelow the plane of the surface for illumination, and curved sides abovethe surface plane of the bottom of the well to allow for the efficientremoval of non-specific light. The sample chambers as shown in FIG. 17,18, and 19 are useful for measuring immobilized samples as well assolution samples.

Practice of the Present Invention to Analytical DiagnosticAssays—Apparatus Types and Test Kits

It is well known in the art that there is a wide range of analyte types.These analytes exist in different sample environments such as water,urine, blood, sputum, tissue, soil, air, and the like. Depending on therequirements of a particular type of analytic assay, one may want to getsemi-quantitative or quantitative information, or both with regard tothe analytes of interest. There are conditions where it is desirable toperform the analysis with a small, inexpensive, and highly portableinstrument. For example, consumer use, use in the field (away from alab), or at bedside in the hospital. One wants to be able to quickly geteither semi-quantitative and/or quantitative measurements on theanalytes in question. In other applications, it is desirable to have asmall and inexpensive instrument for analyte detection in a small labwhere from a few to several samples are tested a day, capable ofquantitative results. For example, doctor's office, clinic, satellitetesting lab, research labs and the like. There are also conditions whereone wants to test several hundred to thousand samples per day, such ashigh throughput testing. Each of the above testing conditions andenvironments thus require different types of apparatus means. Theadvantages and disadvantages in terms of ease of use and cost of suchapparatus can only be determined in detail when the exact requirementsof testing for the analyte(s) in a sample is well defined.

We have determined that the use of certain metal-like particles withcertain variations of the DLASLPD methods of detection allow for thedevelopment of specific test kits and apparatus for the above mentionedtesting environments and applications. There are numerous differentcombinations of analytes, testing environments, sample types, assayformats, analyte detection requirements, and apparatus cost and sizerequirements. One of average skill in the art will recognize thetremendous utility of the present invention in that the practice of thecurrent invention in one form or another leads to easy to use andinexpensive apparatus and test kits to solve most analytical ordiagnostic testing needs.

There are many different configurations and combinations of the DLASLPDmethods, particle types, and sample types that are used together toachieve a specific analyte detection capability. In any specificdiagnostic assay application, the sample type(s), and method(s) ofillumination and detection are usually fixed, as for example, assayformat, sample chamber type, and detection apparatus. The metal-likeparticles have unique scattered light properties which vary by size,shape, composition, and homogeneity of the particles. The specificproperties of light scattering that can be detected and/or measured fromthe particles is set by the previous mentioned particle properties, andthe method and apparatus used to detect and measure the scattered lightproperties. Therefore, the ultimate utility and practice of the currentinvention in one form or another is achieved by combining variousaspects of the illumination and detection means, and sample type, withthe appropriate type(s) of light scattering particles. This results inspecific apparatus and test kits.

One skilled in the art can practice many different aspects of thisinvention by using various particle types, assay formats, and apparatusin many different configurations to achieve many different resultantdiagnostic analytic detection capabilities. FIG. 22 diagrams the variousaspects of the invention which, when configured in a specificcombination, yields apparatus and test kits to suit a specificdiagnostic analytic testing need. The resulting apparatus and test kitsare made by choosing the appropriate components of theMethodology/Apparatus Type Configuration (FIG. 23) and the Particle TypeConfiguration (FIG. 24). FIG. 23 shows that one skilled in the artchooses the illumination source, method and other apparatus components,the assay and sample type, and the detection method and apparatuscomponents. FIG. 24 shows that one skilled in the art chooses theappropriate particle composition, shape, size and homogeneity to detectthe desired light scattering properties of the particle. These processesas outlined in FIGS. 23 and 24, and summarized in the diagram of FIG.22, lead to specific apparatus and test kits. The diagram in FIG. 25shows one of the general methods we have used to develop specificapparatus and test kits for a particular diagnostic testing need. Oneskilled in the art does not need to practice the method in FIG. 25 topractice the present invention in one form or another.

The remarkable signal generation and detection capabilities of combiningmetal-like particles with the DLASLPD methods of illumination anddetection as described herein allows for a wide range of analytedetection sensitivities. With regard to the general types of testingenvironments and the FIGS. 22-25 briefly described above, one skilled inthe art can easily develop apparatus and test kits where in somediagnostic testing applications one only need to use the naked eye fordetection and/or measurement, and in other cases a simple light sourcesuch as an LED (light emitting diode) or low power filament lamp areused and a photodiode or photodiode array can be used to detect and/ormeasure the signal. In other analytical testing applications, a laser,laser diode, filament lamp, or the like can be used with a camera, videocamera or other CCD device (charge-coupled device), and with simpleimage processing means, the scattered light from the particles in amicroarray format, or any other format can be detected and measured.These examples are not meant to be limiting but rather to generally showthe versatility and broad utility of the present invention to detect oneor more analytes of interest in a sample.

For example, a small hand-held apparatus for portable use that canmeasure one or more analytes in a sample can be built by using a lowpower filament bulb, LED, or laser-diode as a light source. A photodiodeor photodiode array is used as a detector. Depending on the sensitivityof detection required, certain types of metal-like particles can be usedwith this apparatus to satisfy the analyte detection requirements. Testkits are constructed for multi-analyte or single analyte detection inliquid or solid-phase samples. For example, for liquid samples,different particle types, each having different easily detectablescattered light properties are used. In solid-phase samples and formats,such as a microarray, one particle type can be used for all thedifferent analytes or various combinations of particle types could beused (depending on the concentrations of the different analytes in thesample).

In another example, an inexpensive apparatus and test kits capable ofmeasuring to low analyte concentrations can be constructed as follows. Alow or high power light source is used with a photomultiplier tube,photodiode array, or video camera. A lens is used to collect thescattered light from the surface(s) containing the particles. Amicroprocessor or external desktop computer is used to collect andanalyze the scattered light data. Test kits for multi-analytesolid-phase analysis are made by using the proper particle type(s) withappropriate microarray sample chambers to achieve the concentrationranges and detection limits as required. This type of apparatus and testkits may be useful in research labs, doctor's offices, satelliteclinics, environmental testing labs, and high throughput testing labs.

The above examples of apparatus and test kits are given as illustrativeexamples and should not be interpreted as the only practices of thepresent invention. One skilled in the art will recognize the broadutility of the present invention. By practicing one or more aspects ofthe present invention to suit a specific analyte(s) detection need, oneskilled in the art will recognize the wide range of apparatus and testkits that can be made.

Assays Involving the Association or Aggregation of Two or More Particlesby Interaction of Analyte and Specific Analyte Recognition Reagents.

It is known in the art that by using the appropriate binding agent(s)and concentration of binding agents and analyte, agglutination,aggregation, cross-linking, networking and similar binding events canoccur and that these events can be used to detect one or more analytesin a sample. In some immunoassays, visible precipitates are formed ifthe antigen is soluble and multivalent while agglutinated or clumpedparticles are formed if the antigen is particulate and multivalent. Insome nucleic acid assays, one specific single-stranded probe can“crosslink” two or more single-stranded targets and propagate networks.Alternatively, two or more different unique single-stranded nucleic acidprobes can be used to bind to different sites on the same targetsingle-stranded nucleic acid. In this approach, cross-linking of two ormore targets can be accomplished, or, by just having the two uniqueprobe sequences bound to the same target allows for detection.

The present invention allows for easier to use, more sensitive, and moreversatile detection of analytes than was previously possible. Inspecific assay formats, the submicroscopic particles of this inventioncan form different types of aggregates that can be detected visually orinstrumentally in a microscope or through macroscopic observation ormeasurements without having to separate free from analyte boundparticles. The type of aggregates formed depends on the size of thecross-linking agent or agents and their valency and on the type ofbinding agent attached to the particle. Aggregates can range from twoparticles to many.

The particles used in a homogeneous, liquid phase, aggregation detectionassay can be labeled directly or indirectly. In a direct labeling assay,an agent that can bind directly to the analyte is attached to the signalgenerating particle. For example, in a direct labeling nucleic acidanalyte assay, the DNA probe is attached to the light scatteringparticle. In an indirect assay, the analyte detecting agent is labeledwith a chemical group A and the particle is labeled or coated with anagent that can recognize the group A. Using direct or indirect labeling,an assay can be formatted so that interaction of the analyte recognitionbinding agents with the analyte (and with group A in the case ofindirect labeling) lead to aggregation of the particles. The aggregatescan be composed of two or more particles.

We have found that if the aggregating or cross-linking agent(s) in anassay are small in size so that the particles in the aggregate are invery close proximity, then aggregates containing two or at most a fewparticles appear as a single particle (submicroscopic aggregate) in themicroscope. However, this submicroscopic aggregate displays differentlight scattering properties than unaggregated particles due toparticle-particle perturbations. Depending on particle composition, sizeand shape, we have observed changes in the color, intensity RIFSLIW, andpolarization of the scattered light. These changes can be used tomeasure the amount of analyte without having to separate free fromanalyte bound particles. In a microscope the changes can be used todistinguish submicroscopic aggregates from nonaggregated particles eventhough both may appear as single particles. If the aggregating orcross-linking agents are large in size, such as a long DNA chain wherethe distance between the particles in the aggregate is larger than theresolution of the microscope, then the particles in the aggregate can beseen individually and distinguished from unaggregated particles by thefact that they stay or move together. These microscopic aggregates canreadily be seen in the microscope when as few as two particles are inthe aggregate. If the distance is sufficiently large so thatparticle-particle perturbations are small, than the particles in theaggregate retain their original light scattering properties. There arealso particle-particle separation distances which are between the twogeneral cases discussed above. We have observed in some specific casesthat the particles in a submicroscopic aggregate do not perturb theirlight scattering properties presumably because they are not close enoughfor particle-particle-perturbation to occur. The aggregate cannevertheless be distinguished from nonaggregated particles because theirintensity is n times that of the unaggregated particle where n is thenumber of particles in an aggregate and/or the particles are “fixed”into position relative to one another.

From the above discussion, it can be seen that liquid, phase homogeneousassays based on macroscopic measurements or visual observations canreadily be achieved if aggregates produced by the presence of analytehave different light scattering properties than free unaggregatedparticles. If particle-particle perturbations in the aggregates aresmall so that the light scattering properties of aggregated and freeparticles are similar, homogeneous assays are still possible usingmethods of detection which allow visualization or measurement of thelight scattering intensities of the individual particles and aggregates.In the situation where the individual particles in an aggregate can beseen, then the aggregates can be easily distinguished from freeparticles and quantified as described above by visual observations orcomputerized image analysis. Aggregates can also be distinguished fromfree particles and quantified in a flow cytometer or similar apparatusor device since aggregates would have a higher light intensity thanindividual particles. In situations where the individual particles in anaggregate cannot be seen in the microscope andparticle-particle-perturbations are absent, the free particles andaggregates can be distinguished by their differences in intensities andthe number of particles in an aggregate established from the scatteredlight intensity of the aggregate (assuming that the intensities areadditional). This can be done by image analysis or flow cytometry andincludes the development of an image by laser scanning or other methodswhich can spatially analyze an area or volume of the sample.

As the number of particles in a submicroscopic aggregate increases, theaggregate can be seen as an enlarged particle or large particle eventhough the individual submicroscopic particles in the aggregate may notvisible through the microscope. In the case of microscopic aggregates,increase in the number of particles in the aggregate produces visiblenetworks and the particles in the network can be counted. Large networksand particle aggregates produce macroscopic entities that can beobserved with the unaided eye and can form precipitates or agglutinates.

One of average skill in the art will recognize that the differentaggregation phenomena described in the preceding paragraph can beexploited to develop many different types of homogeneous assays, some ofwhich employ a microscope or other image analysis techniques and otherswhich involve macroscopic observations or measurements.

The following are selected illustrative examples of how a homogeneoustype or other types of assays can be performed.

Examples of Assay Formats Using Light Scattering Particles

Below are given a few illustrative examples which demonstrate the broadversatility and great utility of the present invention in differentassay formats. One of average skill in the art will recognize that thereare many variations of the present invention which allows for the morespecific, easier to use, and more sensitive detection of one or moreanalytes in a sample than was previously possible.

i. Assay Formats involving the association of two or more particles bymolecular recognition based binding events.

General Principles

In one set of experiments, we biotinylated the surface of a preparationof 40 nm diameter gold particles using the method of base materialmolecule attachment. After purification by centrifugation and washing,we placed a drop of this material on a glass slide and covered it with acoverslip, and observed with the light microscope using the DLASLPDmethod of light illumination and detection. The material appearedhomogeneous, the particles moving very fast in Brownian motion with agreen color. We then removed the coverslip, and placed a drop of asolution of streptavidin onto the slide and recovered it with thecoverslip. After a period of time, new yellow-orange and orange, andorange-white colored particle structures appeared in solution which hadmuch greater intensity and moved much slower than the green particles.Some of these new particle structures also appeared to be asymmetrical,as they flickered as they rotated in solution. After some time, many ofthe green particles had disappeared and there were many of theseyellow-orange and orange particle aggregates. When the edge of thecoverslip was examined under the microscope, it was coated with a layerof orange, yellow-orange, and white-orange particle aggregates whichwere very bright in color. We have observed similar phenomena in ahomopolymer nucleic acid system. These observations show that in variousforms of the present invention, changes in the particle scattered lightproperties can be used to detect molecular binding events either byvisualization of the aggregates, decrease in the number of “free” singleparticles, or in bulk solution by using other methods. For example, fordetection in bulk solution or a flow system, the increase in number ofnew particle forms with unique scattered light properties and/or thedecrease in the amount of particles with the original properties byilluminating a part of the solution under appropriate conditions andlooking for changes in the scattered light emanating from the solution.Alternatively, by using a flow-based system, the material in the samplecan be more specifically analyzed. For example, a microchannel,capillary, or flow cytometry apparatus or device is used such that aportion or the entire sample solution can be analyzed on a particle byparticle basis. The solution flows by an illumination source(s) anddetector(s) or alternatively, the solution is captured in a microchannelor capillary tube and then all or part of the microchannel or tube isanalyzed by moving either the sample container, light source or detector(or some combination of these) along the length of the sample.

For example, a certain nucleic acid analyte is composed of about 100nucleic acid bases and is present in a sample. The sample is prepared sothat this nucleic acid is in single-strand form. Then two or more uniquesingle-stranded “probe” nucleic acid sequences are added to the samplewhere these different probe nucleic acids bind to different regions ofthe target strand. Each of these probe nucleic acids also has attachedto by indirect or direct labeling means one or more particles. Followingincubation, the sample is placed in a flow cytometer apparatus orsimilar flow device where the solution containing the sample can beanalyzed. If the target sequence is present, there will be two or moreparticles which are “bound” together in close proximity. Depending onthe separation distance of the particles, particle-particleperturbations may or may not be occur. These molecular structures whichcontain two or more particles as a result of the hybridization of theprobe strands to the target strand are detected using appropriate meansas described earlier.

ii. Assays Involving the Release of Molecular Entities

There are assay format applications where the present invention can beused to detect the presence of an analyte as the result of a molecular,chemical, or other event. For example, intra or inter molecular bonds,linkages, or other molecular structures can be altered such that theoverall molecular geometry changes, or, a molecular piece(s) isdissociated as a result of the process. For example, peptides, proteins,nucleic acids, or pharmaceutical agents and the like can be attached tothe surface of a sample container by various means that are known in theart. In some of these substances, there is one or more intramolecularlinkage or bonding sites which can be cleaved or otherwise altered bychemical, biological, or other processes. For example, the presence of aspecific enzyme or ribozyme can be detected by monitoring the amount ofcleavage products that are released as a result of it's activity. Alight scattering particle(s) is attached directly or indirectly to areasof the molecular substrate such that the cleavage process is minimallyaffected. The presence and amount of free particles in solution oralternatively, the decrease of bound particles attached to the samplecontainer, or to other particles, can be related to the presence,amount, and activity of the enzyme. In another example, light scatteringparticles have been coated with an antigenic substance and mixed with anantibody such that all of the particles are bound together by theantibody-antigen bond in a multivalent fashion. This network oragglutinated material is placed in or if desired attached to a samplecontainer. A sample is placed into the container which may contain theanalyte (which could be either the same antibody or antigen or acompeting antibody or antigen of somewhat similar structure). Dependingon the presence and amount of antigen or antibody specific analytepresent in the sample, some fraction of the antibodies and particlecoated antigens will become dissociated from the network structure bycompetition. The amount of analyte present can be detected by measuringthe amount of particles in solution and/or the amount of particles thatremain in the agglutinated network. This variation on the method canalso be used by coating the antibodies on the particles, or using otherbinding agents as for example, nucleic acids, peptides, receptors,pharmaceutical agents, hormones and the like.

iii. Detection and Characterization of Molecular Binding Events

In another illustrative example, the Brownian motion of a particle thatis coated with a binding agent can be used in an image analysis formatto detect the presence and amount of an analyte present. This method canalso be used to study the molecular binding event and properties in abinding pair where one partner is attached to the particle and the otheris free in solution. Such capabilities are extremely important incharacterizing the binding properties of antibodies, antigens,pharmaceutical agents, receptors and any substance where it's molecularbinding properties are important. For example, a 40 nm gold particlepreparation is made to contain either antigen, pharmaceutical agent, orantibody on the surface. These particle-binding agents are then placedon a microscope slide and viewed on a microscope using the methods ofDLASLPD illumination and detection. Their Brownian motion properties andquantified. Then a sample solution which may contain an analyte that maybind to the attached binding agent on the particle is added. If theadded solution contains the binding agent partner it will bind to thebound binding agent on the particle and a change in the Brownian motioncan be observed. Alternatively, for characterization applications, knownconcentrations of the substance whose molecular properties are beingcharacterization are titrated in at known concentrations to determineit's binding properties. In this fashion the molecular binding events ofmost any molecular recognition binding pair can be studied.

iv. Amplified Detection of Analytes

In certain analytical and diagnostic assays, it may be preferable toincrease the detectability of the scattered light properties of theparticles so that very simplified or no detection instrumentation isrequired. By use of the appropriate molecular recognition binding-pairsand particles it is possible to significantly increase the level ofdetection sensitivity. Single-stranded homopolymer sequences,avidin-biotin, streptavidin-biotin, and other binding-pair systems canbe used to “chain-together” and “build-up” many particles. As anexample, a solid-phase assay is designed where a sandwichantibody-antigen-antibody structure is formed. One antibody is attachedto a solid-phase so that the antigen analyte is captured. Additionalantibody is then added which contains a biotin group. Then particlesthat are coated with streptavidin and free biotin are added to thesolution. From the (solid-phase-antibody)-antigen-(antibody-biotin)complex grows a . . .(streptavidin-particle)-biotin-(streptavidin-particle)- . . . structurewhich contains many particles bound together. Such a particle aggregateor network structure produces a high level of intensity which is mucheasier to detect than one particle. As another example,polydeoxyadenylic acid (Poly dA) and polythymidylic acid (Poly dT) orother homopolymer single-stranded nucleic acids can be used where PolydA homopolymer sequence is incorporated into a region of thesingle-stranded “probe” molecule. Particles are coated with thecomplementary dT sequence to this homopolymer and are added to thesample with additional “free” dA single-strands to produce the structurecontaining many particles. The above examples are for illustrativepurposes and one of ordinary skill in the art will recognize that thereare many variations of this aspect of the invention possible dependingon the analytical and diagnostic conditions and requirements.

Improved Particle-binding agent Reagents

The attachment of binding agents that are proteinacious such asantibodies to metal-like and non-metal like particles and other surfacesby the method of adsorption are well known in the art (see M.Horisberger, Scanning Electron Microscopy (1981),2, p9-31). The methodof adsorption can be used as for example, with antibody molecules toattach substances which have a binding property to the particle. In thecase of antibodies, the attachment of the antibody molecules to theparticle also confers partial chemical stability to the particle. If theadsorption conditions are carefully controlled, some of the antibodymolecules will still possess binding activity towards it's respectiveantigen. The use of certain synthetic and biological polymers aschemical stabilizers for metal particles is also known in the art (seeHeller et al. (1960), Journal of Polymer Science, 47, p203-217). Themethods of adsorption of substances to particles and other surfaces arehereby incorporated by reference herein.

The exact mechanism(s) and nature of the adsorption of substances toparticles and other surfaces is not well understood. When antibodymolecules are adsorbed to a particle or other surface, the density andorientation of the adsorbed antibodies seem to be related to the levelof binding activity. Due to the lack of control of the adsorptionprocess, it is likely that many of the bound antibody molecules havebecome attached in such a manner that the molecular recognition regionof the molecular structure has been altered such that the bindingactivity may become significantly reduced or possess no activity at all.

While the method of adsorption provides for the attachment ofproteinacious binding agents and other substances which may or may notbe useful in analyte assays to particles, it is difficult to attach sometypes of substances which may be of interest in analyte testing andother fields. For example, nucleic acids, smaller protein andprotein-like substances such as peptides, and other non-proteinacioussubstances such as low molecular weight antigenic substances, hormones,pharmaceutical agents and the like can not effectively be attached toparticles by the adsorption process. Further limitations of theadsorption technique are that there are unique adsorption conditions foreach type of substance which must be carefully controlled. Even whensuch procedures are followed rigorously, there can be significantvariation in the amount of protein and the integrity and bindingproperties of the substance that has been adsorbed to the surface. Inmany cases the binding activity (affinity and specificity) of theadsorbed binding agent is significantly reduced as compared to theunadsorbed form.

Our experience with attaching various proteinacious binding agents suchas antibodies to the surface of the particle by using the method ofadsorption have shown us that there is great variability in the bindingproperties and stability of the resulting particle-binding agentmaterials. The binding affinities of the adsorbed antibodies or otherbinding agents are highly sensitive to the labeling conditions and canalso vary significantly from batch to batch. A significant decrease inthe binding activity of antibodies, avidin, streptavidin and otherbinding agents that have been adsorbed to the particle is common. Insome of the preparations, it appears that some fraction of the adsorbedbinding agent is prone to dissociating from the particle. This can poseserious problems as this dissociated material will compete against theparticle-binding agent for analyte in an analytical or diagnostic assay.

Such lack of control of the attachment process, variability in bindingactivity, and limitations as to what types of substances that can beattached to particles by adsorption methods poses many problems for theproduction and use of such materials for analytic diagnostic purposes.Perhaps most importantly, particle-binding agent conjugates prepared bythe adsorption technique may not be of sufficient quality for manyanalytical applications where very or ultra low concentrations ofanalytes are being detected.

It would be of great use in the art to have a method where any type ofsubstance including binding agents of varying size and composition couldbe attached specifically to a particle or surface whereby the bindingactivity of the attached substance is minimally affected. It would alsobe of great use in the art to have a method for achieving a desireddensity of agent per particle (or, in general any surface). In addition,it would be desirable for these methods to allow binding of more thanone type of agent. From a manufacturing and cost standpoint, it would beof great utility if the synthetic procedures are easy and inexpensive toperform such that a wide variety of different types of substances can beattached to particles using the same basic procedures.

We have developed new methods that allow for the specific attachment ofbinding agents and most other substances to metal-like particles andother surfaces. The particle reagents produced by these new methods arehighly stable and possess high binding affinities with low non-specificbinding properties. These new methods overcome many of the limitationsof the prior art adsorption procedures with the additional benefit thatthe procedures are easy to perform at low cost. In some embodiments,these new procedures allow for a universal linker chemistry platformwhere almost any type of substance can be quickly and simply attached toa particle or surface using many of the same materials and procedures.This is extremely important in the day to day manufacture of suchparticle-binding agent reagents for use in analyte testing.

The following procedures apply to any substance which includes bindingagents or other substances as for example, antigens, antibodies,lectins, carbohydrates, biotin, avidin, streptavidin, nucleic acids,peptides and proteins, receptors, pharmaceutical agents and the like.The methods can be used to attach most substances to metal, metal-like,and some non-metal like particles and macroscopic surfaces. For example,non-metal-like surfaces and particles include materials that may becomposed of organic or inorganic material such as glass, plastics, andthe like.

Methods of Attachment of Substances to Particles and Other Surfaces

i. Base Material Molecule Method

In this method of attaching substances to particles or other surfaces, atwo step approach which involves the use of base material molecules isused. Suitable base material molecules are any substance which canapproach and interact with the surface by adsorption or other chemicalprocess, and have accessible functional groups to which additionalsubstances, as for example, binding agents can be attached. The basematerial molecule may also have the additional property of conferringchemical stability to the particle. Generally the base material moleculeis of a macromolecular form (size >1000 MW) but it can be smaller ormuch larger. Preferred base material molecules are those which attach tothe particle with high affinity, confer some level of physical stabilityto the particle, and possess accessible chemical groups which are easyto conjugate most any substance thereto. The chemical groups allow forbinding agents or other substances to be linked either through chemical,covalent, or non-covalent attachment. For example, covalent attachmentcould involve photochemical or chemical attachment. Non-covalentattachment could involve cross-linking with molecules such asstreptavidin, or by adsorption through hydrophobic, hydrogen bonding, orelectrostatic interactions. The base material molecule may also containone or more chemical groups which can be used to cross-link several baseunit molecules together across the surface of the particle utilizing theappropriate chemical or cross-linking agents.

The following are selected examples of how the method of base materialmolecule attachment can be used to create particle-binding agentreagents which are highly stable, possess high binding affinities forthe entity(ies) they bind to, and provide for a highly flexible, easy touse and low cost method of attaching most any substance to particles orother surfaces. One of ordinary skill in the art will recognize thatthere are many variations of the general technique to synthesizeparticle-binding agent reagents for most any purpose. Using this newmethod, antibodies, peptides, proteins, nucleic acids, pharmaceuticalagents and most any other substance can be attached to the particle in ahighly controlled and predictable fashion.

As an example, we have used a derivative of a polyethylene glycolcompound of approximately MW 20,000. The properties of this molecule(bis(Polyoxyethylene bis[3-Amino-2-hydroxypropyl])) allow for it's useas a base material molecule. Each molecule of this polymer has fouramine groups which can serve as linkage sites for the conjugation ofadditional substances. The hydrophobic backbone of the polyethylenederivative interacts with the particle and is attached to the particlesurface by adsorption or some other process. This interaction is verystrong, as we have not detected any of this material dissociating fromthe particle surface following labeling and use in analytical anddiagnostic assays. The amine groups do not appear to interact with theparticle surface and are accessible as conjugation sites for theattachment of additional substances as for example, binding agents.Using this polymer as the base molecule we have prepared two differenttypes of particle-binding agent reagents. One reagent contains biotingroups as binding agents and the other particle-binding agent reagentwas made to contain single-stranded nucleic acids as binding agents. Thebiotin used for attachment was a chemically modified form where it willcovalently link to amine groups. For the nucleic acids, the 5′ ends werechemically modified so that they would chemically react with the aminegroups. In our use of these reagents in various assay formats we haveobserved that both of these particle-binding agent reagents demonstrateda high degree of stability in low and high salt aqueous solutions withexceptional binding activities. In experiments where the particle-biotinreagent was used no effect upon the binding affinities was detected.This was determined by placing the concentration of the particle-biotinreagent at concentrations of 6×10⁻¹⁴M in suspension and submerging aplastic solid-phase that was coated with avidin into this solution.After a couple of hours of incubation the solid-phase was removed andwashed. When examined under the light microscope using DLASLPD methodsof illumination and detection, particles were detected specificallybound to the avidin coated solid-phase while the control solid-phase(which contains no avidin) showed no particle binding. At these workingconcentrations of particle-biotin reagent, if the binding properties ofthe biotin attached to the particles was substantially decreased, nobinding would have been visible.

In another example, gelatin is used as a base material and the gelatincan be cross-linked on the particle surface by use of chromate ion orother cross-linking agents to minimize the chance of desorption. Bindingagents or other substances are then linked to the particle by using theappropriate conjugation chemistries for attachment of these substancesto accessible amine, carboxylic or other chemical groups to whichattachment can be accomplished.

In another example, streptavidin or avidin can be used as a basematerial. Substances such as binding agents and the like are attached tothe particle by using a chemically modified version of the moleculewhich contains at least one biotin group.

In a further example, polymer-like materials and other materials whichpossess polymer-like properties, as for example, carbohydrates,polyamino acids, proteins, and the like can also be polymerized fromco-polymer units in solution right onto the surface of the particleunder appropriate conditions.

For all of the above examples, one can also first conjugate the bindingagents or other substances to the base material and then apply thismaterial to the particle surface with or without chemicallycross-linking the base materials together. In addition, two or moredifferent types of base material molecules, or one or more base materialmolecules can be used with other chemical stabilizer molecules such thatthe amount of chemically reactive groups available for conjugation andthe chemical stability of the particle-binding agent conjugate can beadjusted to suit most any analytical need.

In the examples above, available materials were used and selected foruse as base material molecules. One skilled in the art can synthesizenew types of base material molecules to further optimize their use forattachment of substances to particles and other surfaces. The followingimprovements allow for particle-binding agent reagents which are morechemically stable, and optimization of the conjugation process withenhanced performance with regard to binding affinities of the attachedbinding agents or other substances. For example, additional chemicalgroups can be added to the backbone structure of the polymer whichincreases the stability of the binding of the base material molecule tothe surface of the particle. Linker arms of various lengths withappropriate reactive chemical groups at the end or close thereto can beadded to increase the distance from the particle at which the bindingagent or other substance is attached and ultimately resides. Differenttypes of reactive chemical groups can be added to the base material tofurther improve the ability to cross-link or otherwise attach theindividual base material molecules together across the surface of theparticle.

ii. Direct Attachment of Substances to Particles or Other Surfaces ByMeans of Chemical Groups Which Adsorb to Metal Surfaces.

We have developed additional methods which allow for direct attachmentof many different types of substances, including binding agents, to beattached to metal and metal-like particles and surfaces. In the art ofmaterial science and related fields, it is known that certain types ofsmall molecules (<1000 MW) can be attached to metal surfaces and thelike. For most of these small molecules there are certain types ofchemical groups at specific locations within the molecule which allowfor one part of the small molecule to become bound to the metal surfacewhile other parts are not bound to the surface. For example, theadsorption of thiol and disulfide containing substances, and amphiphilicsubstances, such as n-alkonic acids and certain detergent molecules ontometal surfaces is known in the art of material science (see Nuzzo et al.(1983), Journal of the American Chemical Society, 105, p4481-4483;Allara et al. (1984), Langmuir, 1, p45-52; and Bain et al. (1989),Journal of the American Chemical Society, 111, p321-335). The methods ofadsorption of substances to metal surfaces are hereby incorporated byreference herein. Therefore, the properties which allow for attachmentof the above substances can be conferred onto binding agents and othersubstances by incorporating the appropriate chemical into specificlocation(s) within the molecular structure of the substance that is tobe attached. Certain types of substances will be easier to attach withthis method than others. For example, substances whose molecularstructure is charged or ionic, or is polarized such that at one end ofthe molecular structure it is hydrophobic while at the other end it ishydrophilic will generally be useful in this particular variation of themethod.

For example, nucleic acids contain a phosphate backbone which contains ahigh negative charge. A single-stranded nucleic acid is end labeled witha thiol or disulfide at the 3′ or 5′ end with or without additionalhydrophobic groups incorporated into the same region of the molecule.This modified nucleic acid will bind to the metal surface or particle atthe end labeled with these groups. The ionic part of the nucleic acidkeeps the main chain of the nucleic acid's molecular structure away fromthe surface such that it is accessible for molecular interactions withmost any substance that can specifically bind to it.

Other substances such as biotin, peptides, pharmaceutical agents,polymers, and the like can be attached to the particle using thismethod. The method is generally useful for most substances which do notinteract significantly with the particle or surface in their nativeform. For substances that may interact with the particle or surfaceadditional methods are required. For example, certain small molecules,proteins and the like may interact with the particle or surface suchthat their binding activity is diminished. In one variation of themethod, the particle is first labeled with, for example, a polymerstabilizing agent. Following this labeling, there are usually open areason the surface of the particle to which small molecule entities canbind. The appropriately modified substance is then added to thechemically stabilized particle to confer a desired binding activity orother property. Alternatively, the chemical stabilizer and chemicallymodified binding agents can be mixed together in a desired ratio priorto mixing with the particle or surface. By using these methods, theamounts and types of substances that are attached to the particle orsurface can be controlled to yield a coated surface or particle with thedesired chemical stability and binding activity properties.

Linker arms of various lengths and composition can also be incorporatedinto the molecular structure. For example, a small molecular weight basematerial molecule an be used where it's molecular structure is optimizedfor attachment to the particle or surface, attachment of most anysubstance to it with any desired orientation, and with a high level ofbinding activity. As an example, a linear polypeptide twenty amino acidsin length is chemically modified at one terminus by the addition ofdisulfide or thiol chemical groups. The native polypeptide is composedof amino acids such that the polypeptide chain will not interact withthe surface except through the chemically modified end. At the otherterminus a free amino group exists, or alternatively, has beenchemically modified for a desired conjugation process such that most anysubstance can be attached at this position. This low molecular weightbase material molecule then is used in one or more variations of themethods as described herein.

The method of base material molecule conjugation and the method ofdirect attachment as described herein allows for the more specificcontrol of the amounts, types, and orientations of substances that canbe attached to particles and other surfaces. A further advantage is thatthese methods provide for the synthesis of particle-binding agentreagents where the binding affinity of the attached binding agentremains at high levels.

An important feature of the base material molecule method utilizingeither small molecular weight or larger molecular weight base materialmolecules is that with the proper selection and utilization of basematerial molecules, the base material molecule can serve as a universallinker platform to which most any substance can be attached to aparticle or surface. This capability becomes extremely important in theday to day manufacturing of particle based reagents for testing ofanalytes. One of ordinary skill in the art will recognize the manydifferent variations of these new attachment methods that can made byvarying the chemical groups, molecular weights, molecular structure,labeling reaction conditions, and the type of conjugation chemistry(i.e. cross-linking, covalent attachment, etc.) that is used.

Microarray or Micropattern Assays with Light Scattering Particles

The microarray or micropattern method of analysis uses discreetspatially addressable areas of a solid-phase to detect different typesof analytes. For example, each spatially addressable area or microspotmay contain a different type of antibody, receptor, nucleic acid or thelike. The arrangement of the spatially addressable areas on thesolid-phase is dictated by the size of the solid-phase, the number ofanalytes or different areas that will be used, and the method ofdetection. Each of the spatially addressable microspots which contain aparticular type of binding agent may be shaped as a square, circle, orany pattern depending on the methods used to make the microarray. Thedimensions may be from a few square microns to square millimeters oreven larger. The microarray method can be implemented using any one ofthe many solid-phase formats that are used for single analyte detectionand in which the final quantification is done by measuring a solid-phasesignal that is related to the amount of analyte bound to thesolid-phase. In practice, the general analytical steps for themicroarray method are as follows. The microarray is exposed to ananalyte sample, e.g. serum, and after a suitable incubation period thearray is washed and exposed to a second analyte binding entity. In oneformat, the second analyte binding entity is bound to the lightscattering particles whose light scattering properties are detected. Thenumber of light scattering particles attached to each microspot is thena measure of the amount of analyte present in each microspot and can becorrelated with the concentration of the analyte in the sample. Inanother format, the second specific analyte binding entity is not boundto the light scattering particles. In this latter format, a thirdentity, that binds specifically to the second specific binding entity,is bound to the light scattering particles. This third entity, forexample, can be streptavidin which binds specifically to biotincovalently attached to the second entity. There are many other assaymethods which can be used for detecting the second entity with the thirdentity bound to the light scattering particles. In any of these formats,the amount of analyte bound to each microspot is established bymeasuring a light scattering signal that is related to the number oflight scattering particles bound to each microspot.

Different methods can be used to detect the number of light scatteringparticles on each microspot in a microarray. The amount of analyte boundto each spot is established from the number of light scatteringparticles attached to each spot in the final assay step. In general,some type of imaging system is needed to separate the light scatteringsignals from the different areas in the array. Many different types offormats can be used for imaging and particle quantification. The methodof choice depends on the precision that is required and the number ofsamples to be analyzed per day. The needed precision can range from lowas in the cases where only a positive or negative type of answer isneeded to very high precision in cases where the amount of analyte hasto be determined with a precision of a few percent. Examples ofdifferent imaging and particle quantification formats are now described.

Special features can be introduced into the microarray for any imagingmethod for example, the chemistry of some of the microspots in the arraycan be formulated to yield a background signal, or the chemistry of someof the microspots in the array can be formulated to serve as calibratingspots containing known amounts of analyte. The signals from these spotscan be used to correct for variations in incident light intensity, lighttransmission between multi-microspot array carriers, light collectionefficiency and photodetector sensitivity from one sample to the next.

Some specific imaging and light-scattering particle quantificationmethods for applications to microarray and array chips are nowdescribed.

a. The DLASLPD Method with Simple Light Microscope

i. Low particle surface density (less than 0.1 particles per μ²) on aspot. If the number of samples to be examined is not high, the number ofparticles in each spot can be determined by visual or other countingmethods of the number of particles on each spot. A background count isalso made. The counting can be done on liquid covered or drymicroarrays. The number of particles per microspot which is consideredto be positive is defined by previous test experiments. If many samplesare to be examined, the counting can be done automatically using asimple video detector and object counting software.

ii. High particle surface density (greater than 0.1 particles per μ²) ona spot. For positive or negative types of analysis, the intensity fromeach spot can be detected by visual observation or photodetection. AResult is positive if the intensity is higher than that of thebackground. If a quantitative result is needed and there are not toomany samples to be examined (for example, bedside, field, small clinic,or research lab testing), a manual technique with a two observation portmicroscope can be used as follows. A single microspot is illuminatedwith a narrow beam of light. The beam is positioned on the spot byvisual observation through one observation port and the intensity ismeasured quantitatively through a photosensitive device with or withoutspatial filtering aperture depending on the level of stray light signal.The scattered light intensity from each spot is measured by manuallyscanning each spot through the beam. Alternatively, the beam could bescanned manually and the light detected from each spot detected by alarge area photodetector or a small area detector in which the detectorarea is kept confocal with the illuminating spot. This could also beautomated. If many samples are to be analyzed, the microarray can beilluminated with a broad beam of light and an image of the microspotarray digitized through a video camera and frame grabber. The intensityof each microspot is then determined by software image analysis. We havedetermined that these methods allow for very sensitive and wideconcentrations of one or more analytes in a sample to be detected. Oneskilled in the art will appreciate that many other variations of themethod are possible.

Use of Certain Types of Metal-like Particles in Microarray and ArrayChip Detection of Analytes

In our work with microarrays, we have found that metal-like particlesare preferred light scattering particles. The size, shape, composition,and homogeneity of the particle type(s) used in a specific microarrayapplication depend mainly on the following; the amount of non-specificlight background in the sample; if the microarray is dry or covered withliquid; the dimensions of the discreet solid-phase binding areas; amountand concentration ranges of the analyte(s) that are detected; detectionby eye or by photodetector, and measurement by particle counting and/orintensity measurements.

As an example, we were easily able to detect the binding of individual60 nm diameter gold particles coated with BSA-biotin to 80 microndiameter spots containing streptavidin on a plastic solid-phase in amicroarray format covered with buffer solution. We used our customilluminator under DLASLPD conditions and the inexpensive microscopesystem we developed. In microarray streptavidin microspots with lowerdensities of bound 60 nm diameter gold particles coated with BSA-biotin,we counted the number of particles bound. At higher densities, wemeasured the intensity of the scattered light arising from the particlesbound to the individual streptavidin microspots. We detected particledensities down to about 0.06 particles per μ² at a signal to backgroundratio of 13. This implies that for this type of assay, densities down toabout 0.015 particles per μ² can be detected at a signal to backgroundratio of about 3. Very high densities of bound particles were alsodetected (saturation of available binding sites per individual microspotof 80 micron diameter). To perform the same type of microarray assay ina dry form (not covered with liquid), the use of larger diameter goldparticles or other metal-like particles with greater light scatteringpowers may be required to achieve the same sensitivity. Also, using alonger wavelength light source such as a HeNe laser with illuminationabove 600 nm and spatial filtering may also be useful.

For detection of samples by use of a small handheld or other type ofportable device, even larger particles may be needed to be useddepending on the level of sensitivity required as typically one skilledin the art must use low power light sources in such a device.

For multi-analyte detection in the microarray format, the concentrationsof different analytes may exist at very different levels withdifferences of 1,000 to 1,000,000 or even greater in concentration. Insuch situations, the light scattering power and the relative size of theparticles become very important. For example, if one is analyzingmulti-analytes on an array chip or microarray where the individualdiscreet binding areas are about 100 square microns, the number ofparticles that can be bound to this 100 micron square area is highlydependent on the size of the particle used. For example, if a 40 nmparticle is used, at binding saturation, about 79,600 particles can bebound to this area. However, if a particle of 120 nm is used, only about8,800 particles can be bound to the area. Depending on the amount ofnon-specific light background and non-specific binding of the particles,the minimum number of particles that must be bound to the area for areliable measurement can be quite variable. For example, in certainsituations a few thousand or more particles may be needed to be bound tothe binding site area on the microarray in order to get a positivedetection result. Using larger particles thus limits the detectabilityof the analyte. In binding site areas of small dimensions, the smallestparticle that can be used which gives adequate signal/background shouldbe used. In addition, optical and spatial filtering, confocal imaging,more powerful light sources and other instrumental components can beoptimized to increase the detection limit. Similarly, if two or more ofthe analytes exist at very different concentrations, then differenttypes of particles with the appropriate size and light scattering powermay be needed to be used.

These examples are not meant to be limiting but to show how in variousapplications, the selection of certain types of metal-like particlesleads to specific test kits for microarray analysis and detection ofmulti-analytes. One skilled in the art will recognize that there aremany other variations of the method of the present invention to detectmulti-analytes on array chips and microarrays.

Use of Certain Aspects of the Present Invention with other Illuminationand Detection Methods

This discovery means that you can use various aspects of the presentinvention in the art existing diagnostic detection methods and apparatuseven without using the optimal light and detection methods and systemsas the present invention has disclosed. For example, laser confocalmicroscopy methods, brightfield and epi-illumination microscopy methods,and the methods of reflection contrast and differential interferencecontrast microscopy can be used with certain types of metal-likeparticles for measurement of multiple analytes on microarray chips andthe like.

For example, a confocal microscope as described by Ekins in U.S. Pat.No. 5,432,099 (hereby incorporated by reference) can be used. Generally,such confocal microscopy relies on point illumination rather than fieldillumination and usually works in a fluorescence mode withepi-illumination. Usually the detector is a photomultiplier because thesignal to be detected is very low. The light source is frequently alaser. In the present invention even though the signal is extremely high(compared to normal confocal microscopy) and the light source need notbe a laser, an apparatus as complex as the confocal microscope can stillbe used. Clearly, the use of such an apparatus provides even moresensitive detection of particles as described in this invention as wehave found, and minimizes stray light problems.

Thus, in another example, the methodology of Fodor et al. in 364 Nature555, 1993 for detection of target molecules using biological chips canalso be used.

These methods when combined with the use of one or more aspects of thepresent invention are useful in certain microarray analysis applicationswhere the cost and ease of use of the method and apparatus are not ofconcern. We are not aware of anyone using these above mentioned artexisting techniques with metal-like particles and/or the method ofrefractive index enhancement and/or autometallography and/or any otheraspects of the current invention. We thus claim use of these previouslydescribed art existing detection methods and apparatus with one or moreaspects of the present invention.

Other Adaptations of the Invention for Applications to Microarrays

The methods of the present invention provide an excellent means fordetection of one or more analytes using a microarray format. Thefollowing methods provide additional variations which are useful incertain analytical applications.

We can miniaturize our illumination and detection methods such that asingle or multi-optical fiber based apparatus is constructed. Thisprovides an alternative to using the imaging methods for detection.

One problem in using a two-dimensional array, or other type ofsolid-phase spatially addressable system is the problem of signalcrosstalk between different areas of the microarray. Crosstalk, glare orother similar problems can arise from several sources, for example, (1)the individual areas containing the light scattering or fluorescentmaterial(s) are so close together that they appear as one area, or (2)one area contains a high amount of light scattering particles orfluorescence while other adjacent areas contain very low amounts oflight scattering particles or fluorescent materials. Depending on howclose the areas are to each other, some portion of light coming from thehighly intense areas will be picked up by the detector in the areas fromwhich lower light intensities are coming from.

One potential solution in the art is to illuminate each spatiallyaddressable solid-phase site separately by using a scanning process andrecording the light signals coming from each spatially addressable siteseparately when it is illuminated. This can be accomplished by scanningthe different areas one at a time by moving the illumination beam, orthe sample. However, these scanning mechanisms are usually complicatedand add a tremendous amount of cost and complicated procedures to theanalytical method, and may be too costly and not robust enough for theeveryday rigors of a clinical testing laboratory or highly activeresearch laboratory.

An example of a further variation of the present invention is nowdescribed. An optical fiber at one end is beveled to a suitable angleand is used as a discreet illumination source such that when broughtclose to an area to be measured, the emitted fluorescent or scatteredlight from the area is detected from the other side of the samplesurface. This configuration allows for the specific illumination of anarea and eliminates the above mentioned problems of crosstalk. It alsoremoves the requirement for an imaging-type of detector such as a videocamera, and any type of photodetector can be used. As an example, for anarray of 24 microspots or distinct areas to be measured, 24 individualillumination fibers are used, one for each spot. All that is required isthat the individual spots are illuminated at different times withrespect to one another. Several small spatially addressable areas, downto about ½ the diameter of the optical fiber can be measured in thisfashion.

In another embodiment of the method, where the use of epi-illuminationor similar methods are desired, as for example, confocal imaging, onecould miniaturize the system by placing at the end on an optical fiber avery small imaging lens and then achieve confocal conditions where thescattered or fluorescence light can be measured from the desired regionof the microarray. For any area to be measured on the microarraysurface, a single optical fiber is used with a microlens to deliver theincident light and collect the emitted fluorescent or scattered lightthat is to be detected. Multiple optical fibers could be used asdescribed above if desired to detect more than one area of the surfaceat a time.

One of skill in the art will recognize that the preceding illustrativeexamples are only a few of the many variations of the present inventionthat are possible.

Screening of combinatorial synthesized molecule libraries

A tremendous problem currently in the burgeoning field of combinatorialsynthesis of important molecules is the lack of highly sensitive,practical, and easy to use signal and detection technology and assayformats to detect the few copies of newly synthesized combinatorialmolecule(s).

We have determined that our signal and detection technology is easilyused on solid-phases that contain spatially addressable sites such as2-dimensional arrays or any spatially addressable solid-phase.Therefore, our methods in one form or another are directly applicable tothe screening and detection of one or more than one class ofcombinatorial or biocombinatorial molecule(s) in this type of format.The assay method can be any of the known procedures in the art.

The invention as we have described herein can also be used to detect andquantify one or more specific combinatorial, biocombinatorial, orotherwise synthesized molecules on a spatially addressable solid-phase.For example, it is well known in the art that a wide diversity ofbiocombinatorial and combinatorial molecules can be synthesized by usingthe methods of “split synthesis”, “parallel synthesis”, and relatedmethods (all of these methods are incorporated herein). Typically, awide diversity of combinatorial molecules are synthesized on smallparticles or other pieces of solid substrate where each particle orsubstrate piece contains one unique set of combinatorial synthesizedmolecules. There are problems of identifying and purifying those piecesor particles that contain the “active” sets of synthesized molecules inthe art.

There are several ways to utilize our signal and detection system todetect these specific and desirable combinatorial product(s). In oneassay method, a binding agent (that is specific for the desired analyte)is coated on a selected type of metal-like particle. When the coatedparticle is added to the sample, it binds to the analyte. Alternatively,an indirect method involving the use of biotin labeled binding agentfirst binds the analyte, which is then detected by adding streptavidincoated metal-like particles prior to detection. The light scatteringparticle is bound in one form or another to the desired analyte ofinterest which resides on the synthetic solid phase. In this manner, thedesired molecules are identified, isolated, and purified from the sampleby filtration, centrifugation, dialysis or any other art knowntechnique. Alternatively, binding agents labeled with particles coatedwith binding agents can be added such that aggregates or networks areformed between the specific molecule-containing synthetic particles andthe metal-like particles. Similar means as described above are used toidentify and purify the desired molecules.

Multi-analyte analysis for different combinatorial synthesized moleculesis accomplished by using two or more types of metal-like particles eachcoated with a different type of binding agent. Refractive indexenhancement and DLASLPD video-enhanced contrast methods can also beused.

In another assay method, the metal-like particles also contain acomposition of a ferro-electric or magnetic composition such that theseparticles can be manipulated in three dimensional space by using anapplied EMF to the reaction container. In this manner, the substrateparticles containing the “active” combinatorial molecules can be easilypurified and detected from all the other material. It should be notedthat a mixed composition of ferro-electric or magnetic and othermetal-like compositions of specific particles are also very useful inmany other fields including diagnostic assays, and for isolation andpurification of desired molecules. The use of refractive indexenhancement methods in combination with the above methods increases thesensitivity of detection.

Metal-like Particles Used as Solid-Phase Synthetic Supports

Metal like particles when coated with appropriate substances, areexcellent substrates for conducting chemical or biochemical synthesis asfor example, combinatorial synthesis. The specific coating of metal-likeparticles can be made to consist of for example, polymers, antibodies,proteins, nucleic acids, amino acids, reactive chemical groups, and thelike. For example, metal-like particles are coated with a polyethyleneglycol compound containing chemically reactive amine groups. Synthesisis then initiated on these amine groups which are numerous on thesurface of metal-like coated particle. Other reactive chemical groups orgroups that can be specifically activated can be used instead of theamine group. In another example, amino acids, or small peptides arecoated directly on the surface of the metal or metal-like particle, orare chemically linked to polymer or other type of macromolecules thatare coated on the surface of the metal-like particle. Synthesis is theninitiated on the metal-like coated particles. In yet another example,reactive groups are attached to the surface of the metal-like particleso that protein, nucleic acid, chemical, or biochemical synthesis can beperformed. The number of reactive groups on the surface of the particlecan be also modified as follows. A mixture of polyethylene glycolcompounds (MW 20,000) with and without reactive amine groups (or otherreactive groups) are mixed in an appropriate ratio to achieve a desirednumber of reactive groups on the surface per particle. In this manner,the metal-like particle is coated with a specific amount of chemicalsynthetic sites, or binding sites per metal-like particle. The specificnumber of sites and the type of reactive groups sites can be varied tosuit any particular need as for example, further chemical synthesis orfor diagnostic reagent purposes. For example, for diagnostic-typeapplications, adding a discreet number of specific binding agentmolecules per metal-like particle may be important to achieve thedesired assay performance. In addition, two or more different types ofreactive synthetic or binding sites can be placed on the same metal-likeparticle in specific amounts utilizing the same approach as describedfor the polyethylene compound above by mixing in appropriate ratios thedesired substances (i.e. different binding agents or chemical groups,etc.) These types of coated particles may be useful for example, toisolate, purify and detect two or more different molecules using thesame particle. The high densities (grams/cm³) of many types ofmetal-like particles also offers many advantages in the purification,isolation, and identification of molecules of interest. MLSP typeparticles offer the further advantage of more easily manipulating theparticles within the medium. The above examples are only a few of themany possible variations of this method. Other variations will beapparent to one skilled in the art.

Practice of Various Aspects of the Invention Outside the Field ofAnalytical Diagnostics

The present invention features methods to detect one or more analytes ina sample by detection of the scattered light properties of a particle.It should be noted that various aspects of the invention as disclosedherein are directly applicable to many other specific applicationsoutside of the diagnostic field. One skilled in the art or the art ofother fields such as optical information and storage, image formationand processing, electrical-optical signal transduction and switches,telecommunications, information transducers, and many other relatedapplications has been enabled by this disclosure to practice variousaspects of the present invention specifically to solve problems andcreate new products in fields outside of analytic diagnostic assays.

One aspect of the current invention that is very useful for applicationsto other fields is the ability to identify specific metal-like particlesof certain size, shape, composition, and homogeneity by a unique opticalsignature which is characteristic of that type of particle. Embodyingsuch specific optical signatures in a very small structure allows forthese particle signal agents to be used in numerous fields. For example,they can be used in industrial quality control, markers or labels toidentify or trace any product, material, substance, or object thatcontains the particle. The particles in one form or another can be usedas a means for identification and the like similar to the art knownmethod of “barcoding”. For example, a coating containing one or moretypes of particles can be applied to consumer products to identify theauthenticity, date, or other relevant information. Similarly, papercurrency, stock and bond certificates and the like can have coated onthe surface or embedded within the paper material itself certainparticle types that can be detected to determine the authenticity of theobject. Other examples include placing small amounts of a specific typeof particle inside prescription or over the counter drugs toauthenticate or trace of drug. In addition, the particles can be used asenvironmental, industrial, pharmaceutical, or biological tracers tostudy the physical properties of a system such as disposition of fluids,materials, and the like. One skilled in the art will recognize thatthese are just a few of the many possibilities.

Another aspect of the current invention which is directly applicable toother fields is the use of light scattering particles which can bephysically manipulated by electric, magnetic, or related fields. We namesuch particles Manipulatable Light Scattering Particles (MLSP) and theseare described in detail later. Such MLSP particles can be oriented intovarious arrangements in one-, two-, or three-dimensional space by usinga magnetic, electric, or related electromagnetic field (EMF). In thisway the unique light scattering properties of the particles can be usedto form certain patterns, images, or colors. The specific orientationsof one or more MLSP particles are used to store or transduce informationvia the light scattering properties of an individual particle, or theresulting light scattering information, that is, optical signature oftwo or more particles arranged in a particular orientation. For example,three different types of particles that scatter blue, red, and greenlight are placed inside a small area or volume such as a “pixel” in ascreen that contains a specified number of pixels in a two-dimensionalarray. The screen forms a color or black and white image, or movingpicture that is similar to the appearance of a television image, videoimage, motion picture image and the like when the screen is illuminatedwith white light. Each pixel or groups of pixels is spatiallyaddressable by an electric or magnetic field (EMF) such that by applyingthe appropriate EMF, the individual particles that scatter blue, red,and green light are oriented to produce a specific color with a certainhue and intensity when appropriately illuminated. For example, at oneapplied EMF, the red and green particles are concentrated to a very tinyspot while the blue scattering particles are freely dispersed within theinner volume of the pixel. This pixel will then appear blue. A differentEMF can then be applied to cause the same effect for the red or greenlight scattering particles. In this way, by specifically orienting thedifferent particles in each pixel, the desired color image is produced.This method and apparatus offers many attractive advantages to thecurrent cathode ray tube based image formation technology and the like.

In another example, MLSP particles are switched from one specificorientation to another by appropriately adjusting the EMF. For example,asymmetrical silver particles which can produce green or red scatteredlight and/or also have two different levels of intensity of scatteredlight are used as follows. One or more particles are placed in aspecific location in either a liquid type or solid type of materialwhere the particle is able to rotate, that is, re-orient itself an EMFfield is applied to the material or device containing the particles.Depending on how many particles are used and the desired function of thedevice, the different orientations of the particles will signifydifferent types of information. For example, in one orientation, thelight scattering properties of the asymmetric MLSP particle(s) indicatethe “off” or number 0 in a binary code system, while in a differentposition or orientation the light scattering properties indicate the“on” or number 1 in a binary numeric system. The orientation of theasymmetric MLSP particles is changed by varying the EMF to achieve thedesired orientation of the particles in the material or device. Whenlight interacts with the particles in a specific orientation, theproperties of the scattered light signify a specific type of informationas described above. In this manner, simple and multi-component opticalswitches that are useful in telecommunications and related fields can bemade. Similarly, a series of these switches can be assembled in a serialor parallel fashion for more complex information storage and handling.

New types of information storage devices can be made by encrypting orstoring the information by using different types and/or orientations oflight scattering particles and MLSP particles. For example, an opticalstorage disk can be made that is similar to what is known in the art asa “Compact Disk” or “CD-ROM” Disk or the like. Instead of using bumpswhich project above the surface to encode the information, lightscattering particles are used. The particles can be placed on or in anymaterial from which the light scattering properties of the particles canbe detected. In this manner much more specific information and storageof higher densities of information are possible.

One skilled in the art will recognize the many different types ofdevices that can be built using various light scattering particles in aparticular application. The above examples are just a few of the manyways such metal-like and MLSP particles are used outside the field ofanalytic and diagnostic detection. These applications are enabled by thedisclosure herein and applicant hereby claims right to the practice ofvarious parts of the invention as described herein to fields outside thefield of diagnostic analytic assays.

Description of the SpectraMetrix Spectrophotometer Instrument for LiquidSamples and Theory of Operation

The SpectraMetrix Photometer is a right angle photometer that measuresthe intensity of light which is scattered or emitted light at rightangles to the exciting light beam. A schematic diagram of the instrumentis given in FIG. 21. The light source is a microscope illuminator or anyother type of light source. The instrument can be used with or withoutthe monochromator. Adapters for connecting different light sources areprovided with the photometer. Scattered or emitted light is detected bya photomultiplier (PM) tube. The photometer has a manual light shutterto keep light from reaching the PM tube while changing samples. Opticalfilters or polarizers are introduced in the incident or emitted lightpath as required. Cylindrical cuvets (e.g. test tubes) of differentdiameters are used as sample cuvets. However, any type of opticallytransmissive sample container may also be used with the appropriateholder. Test tubes of a diameter of 6 mm by 50 mm were used and amicroscope illuminator with an infrared (heat) filter were used toobtain the data reported herein.

The illuminator can be connected directly to the spectrophotometer orthrough a monochromator. The monochromator used for the measurementsreported herein is a diffraction grating monochromator. The illuminatoris powered by a regulated DC power supply at 6 V and 3 Amps.

In this paragraph we describe the optics of the instrument that we usedwithout monochromator operation. A x10 objective focuses the light fromthe illuminator unto the sample tube. A light collecting lens (23 mmfocal length, 19 mm diameter), that is positioned at right angles to theexciting light beam (about 1.5 inches from center of sample cuvet),focuses an image of the sample tube at a distance of about 106 mm fromthe center of the sample tube. This distance allows a light shutter andfilter holder to be placed between the collecting lens and the PM tube.A diaphragm with a 3.25 mm diameter hole (made with a #20 drill bit) isplaced at the image plane. The PM tube is placed behind the diaphragm.The diaphragm blocks the light reflected from the walls of the cuvet andallows only light scattered from the center of the sample volume toreach the PM tube. The diaphragm, while reducing the amount of scatteredlight that reaches the PM tube, maximizes the signal to backgroundratio. In order to further minimize the detection of light reflectedfrom the sample tube, the tube is positioned at an angle of about 40-50degrees with respect to the vertical direction so that reflected lightdoes not reach the collecting lens. Because of this angle and refractiveindex effects, the light emerging from the tube does not travel alongthe center axis of the collecting lens and the scattered light beam atthe image plane is displaced downward from the center axis of thecollecting lens. This requires that the 3.25 mm aperture and PM tube bedisplaced downwards from the collecting lens axis. The instrument isconstructed such that the downward displacement can be manually adjustedfor the most efficient scattered light detection.

When the monochromator is used, the optics are the same as above exceptthat an additional lens (23 mm focal length, 19 mm diameter) ispositioned between the 10x objective and the monochromator exit slit.The lens is 4 inches from the center of the sample cuvet. The exit slitof the monochromator is 5.6 inches from center of sample cuvet. Theilluminator is connected to the adapter at the entrance slit of themonochromator.

Adjustment of the photometer optics

a. Place a 60 nm, 4×10⁻¹² M gold sol in a 6×50 mm culture tube in thesample holder of the spectrometer. Adjust the angle of the tube withrespect to the vertical so that it is between 40 and 50 degrees.Position the angled tube so that the focused exciting light beam crossesthrough the center of the cuvet. Do not allow the exciting light tostrike the front surface of the tube (surface towards collecting lens)as this will increase the amount of light reflected towards thedetecting system.

b. The distance of the collecting lens from the center of the sampletube is adjusted so as to form a sharp image of the walls of the tube ata distance of 106 mm from the tube center. The image of the scatteredlight beam and the walls of the sample tube can be seen clearly on apiece of white paper placed at the distance of about 106 mm from thetube center. The tube image has a diameter of about 8 to 10 mm at theimage plane. The lens should be positioned so as to obtain a sharp imageof the walls of the cuvet. The scattered light beam appears a littlefuzzy at the image plane because of its finite width. The best positionof the lens is about 1.5 inches from the center of the sample cuvet. Theexciting beam can clearly be seen as it crosses a scattering solution.

c. The above adjustments of the collecting lens position are performedwith the structure that contains the shutter, filter holder anddiaphragm holder removed from the instrument. After the lens iscorrectly positioned, the latter structure is replaced and the lightblocking diaphragm with 3.25 mm opening is inserted. The PM tube isinserted into place.

d. After steps a, b, and c, the position of the aperture with respect tothe collecting lens optics is adjusted as follows. Place a piece ofwhite paper in the place where plane of the PM photocathode will bepositioned when the PM is inserted. With light scattering gold particlesin the sample compartment, adjust the position of the aperture until themaximum amount of light on the PM tube is seen. When the aperture isproperly positioned, the light on the paper appears as a 0.32 inch (8mm) diameter spot.

EXAMPLES

Examples 1 through 10 involve the measurement of light scattered fromparticles, or emitted from fluorescent molecules, or both. Theinstrument used to measure the light signal was a photometer built bySpectraMetrix as described previously.

For examples 1 through 3 the polystyrene particles used for thesemeasurements were NIST traceable certified nanospheres obtained fromDuke Scientific Corp., Palo Alto, Calif. The gold particles wereobtained from Goldmark Biologicals, Phillipsburg, N.J., a distributorfor British Biocell Intl., Cardiff UK.

For examples 4 through 10 the fluorescein was obtained from MolecularProbes Inc., Eugene Oregon, the gold particles were obtained fromGoldmark Biologicals, Phillipsburg, N.J., a distributor for BritishBiocell Intl., Cardiff UK., and the Polystyrene particles were obtainedfrom Interfacial Dynamics Inc., Portland, Oreg.

The relative light scattering powers of particles of the same shape andsize, but of different composition, can be directly compared bycomparing the light scattering intensities at right angles to the pathof the incident light. If the light scattering intensity measurement fora known concentration of each particle of interest is done at the rightangle of observation, the light scattering intensities for identicalconcentrations of particles of the same size and shape but of differentcomposition, can be directly compared and the relative total lightscattering powers of the different particles determined.

Examples 1, 2, and 3

Calculated and Measured Relative Scattering Power of ComparablePolystyrene and Gold Particles

The results are presented in Tables 6,7, and 8. Calculations wereperformed using known light scattering relationships and our newlydefined relationships as previously described. Experimental measurementswere done for particles in water by detection of the light scattered byparticles free in solution at a given illumination intensity andwavelength using the SpectraMetrix Photometer. The following steps wereperformed.

(a) Illuminate the control and comparable sized particle samples withthe same incident light composition and intensity.

(b) Determine the light signal emitted from a control tube containingwater but no particles.

(c) Determine the light signal emitted from a tube containing particlesat known concentration.

(d) Subtract the control light signal value (b) from the light signalvalue of (c).

(e) Compare light signals from equal concentration of gold andpolystyrene particles.

Example 4

Measured Relative Signal Generating Power of Fluorescein and GoldParticles—White Light Illumination.

The results are shown in Table 10. The same method of light detectionwas practiced to determine the light signal emitted from all samples ina 6 mm by 50 mm glass tube. No optical filters were used in themeasurement of the light signal from either the gold particles or thefluorescein.

All measurements were made in water. The solution containing thefluorescein had a pH of 8-9. The light signal value of a tube containingonly water was subtracted from the gold particle or fluorescein value inorder to obtain the light signal due to just the fluorescein or goldparticles.

The following steps were performed for the measurement of the lightsignal from particles.

A. (a) Illuminate all samples with the same incident light compositionand intensity.

(b) Determine the light signal emitted from a control tube containingwater but no particles.

(c) Determine the light signal emitted from a tube containing particlesat known concentration.

(d) Subtract the control light signal value (b) from the light signalvalue of (c).

The following steps were performed for the measurement of the lightsignal from fluorescein.

B. (a) Illuminate the samples with incident light of the same intensityand composition as above in

(b) Determine the light signal emitted from the control tube.

(c) Determine the light signal emitted form a known concentration offluorescein in a tube.

(d) Subtract the control light signal value (b) form the light signalvalue of (c).

C. (a) Compare light signals obtained from known concentrations ofparticles and fluorescein molecules.

Example 5

Measured Relative Signal Generating Power of Fluorescein and GoldParticles-Monochromatic Illumination

The results are given in Table 11. These results have not been correctedfor differences in incident light intensity. Monochromatic incidentlight at wavelengths where maximum light emission occurs fromfluorescein (490 nm) and where maximum light scattering occurs form thegold particles was used. The incident light intensity at 490 nm wasslightly lower than the intensities used for the gold particles andranged from about 86 percent of the intensity at 520 nm to about 80percent of the intensity used at 565 nm. On the other hand, the quantumefficiency of the photomultiplier tube ranged from 0.34 at the primaryemission wavelength of fluorescein (520 nm) while it was about 0.18 at560 nm.

Except for the incident wavelength, the same method of light detectionwas used on all samples in a 6 mm by 50 mm glass tube. No opticalfilters were used in the measurement of the light signal from either thegold particles or the fluorescein.

All measurements were made in water. The solution containing thefluorescein had a pH of 8-9. The light signal value of a tube containingonly water was subtracted the gold particle or fluorescein value inorder to obtain the light signal due to just the fluorescein or goldparticles.

The following steps were performed for the measurement of the lightsignal from particles.

A. (a) Determine the light signal emitted from a control tube containingwater but no particles.

(b) the light signal emitted from a tube containing particles at knownconcentration.

(c) Subtract the control light signal value (a) from the light signalvalue of (b).

The following steps were performed for the measurement of the lightsignal from fluorescein.

B. (a) Determine the light signal emitted from the control tube.

(b) Determine the light signal emitted form a known concentration offluorescein in a tube.

(c) Subtract the control light signal value (a) from the light signalvalue of (b).

C. (a) Compare light signals obtained form known concentrations ofparticle sand fluorescein molecules.

Example 6

Measured Relative Signal Generating Power of Fluorescein, Polystyrene,Polystyrene-fluorescent Compound, and Gold Particles

The results are given in Table 12. These results have not been correctedfor differences in incident light intensity.

All samples were illuminated with monochromatic incident light. The 100nm diameter gold particle was illuminated with incident monochromaticlight of a wavelength near where maximum light scattering form theparticle occurs. The polystyrene-fluorescent compound particle samplewas illuminated with monochromatic incident light of a wavelength wheremaximum fluorescence excitation occurred (490 nm). The maximumfluorescence emission for this fluorescent compound occurred at 515 nm.The incident light intensity at 490 nm was about 80 percent of that at555 nm. The quantum efficiency of the photomultiplier tube at 555 nm wasabout 60 percent of that at 515 nm.

Except for the incident wavelength, the same method of light detectionwas used on all samples in 6 mm by 50 mm glass tubes. No optical filterswere used in the measurement of the light signal from either the goldparticles or the fluorescent particles. All measurements were made inwater. The light signal value of a tube containing only water wassubtracted form the gold particle or polystyrene particle value in orderto obtain the light signal due to just the polystyrene or goldparticles. The following steps were performed for the measurement of thelight signal from particles.

A. (a) Determine the light signal emitted from a control tube containingwater but no particles.

(b) Determine the light signal emitted from a tube containing particlesat known concentration.

(c) Subtract the control light signal value (a) from the light signalvalue of (b).

The following steps were performed for the measurement of the lightsignal from the fluorescent particles.

B. (a) Determine the light signal emitted form the control tube.

(b) Determine the light signal emitted from a known concentration offluorescent particles in a tube.

(c) Subtract the control light signal value (a) from the light signalvalue of (b).

C. (a) Compare light signals obtained from known concentrations ofparticles.

Example 7

Detection of 59.6 nm Diameter Gold Particles and Fluorescein at HighSerum Concentration

The results are given in Table 17. The serum was obtained fromBiowhittaker Inc., Walkerville, Md. The serum had been filtered througha one micron filter before sale and was clear and straw colored inappearance. For fluorescein measurements the serum was adjusted to aboutpH 9 to 9.5. The solution containing the gold particles was illuminatedwith monochromatic incident light of 543 nm wavelength, a wavelengthnear that where maximal light scattering from the particle occurs. Thesolution containing the fluorescein was illuminated at 490 nm where themaximal fluorescence excitation occurs.

Except for the incident wavelength, the same method of light detectionwas used for all samples in 6 mm by 50 mm glass tubes. No opticalfilters were used in the measurement of the light signal from either thegold particles or the fluorescein.

Measurements were made in the stated concentration of serum. The lightsignal value of a tube containing only serum at the proper concentrationwas subtracted from the gold particle or fluorescein value in order toobtain the light signal due to just the fluorescein or gold particles.

The following steps were performed for the measurement of the lightsignal from particles.

A. (a) Determine the light signal emitted from a control tube containingserum at the proper concentration but no particles.

(b) Determine the light signal emitted from a tube containing particlesat known concentration.

(c) Subtract the control light signal value (a) from the light signalvalue of (b).

The following steps were performed for the measurement of the lightsignal from the fluorescent solution.

B. (a) Determine the light signal emitted from the control tube.

(b) Determine the light signal emitted from a known concentration offluorescein in a tube.

(c) Subtract the control light signal value (a) from the light signalvalue of (b).

C. (a) Compare light signals obtained from known concentrations ofparticles.

Example 8

Lower Limit of Detection of Fluorescein, Gold and Polystyrene Particlesat 92.8% Serum Concentration

The results are given in Table 18. For the fluorescein measurement, thelight signal emitted from the sample containing fluorescein was passedthrough a Kodak No. 16 Wratten filter before encountering thephotomultiplier tube. The maximum light intensity from the fluoresceinsolution was observed at an incident monochromatic wavelength of 498 nm,while the maximum light scattering from the gold particles was observedat 554 nm. No optical filters were used in the measurement of the lightsignal from the gold or polystyrene particles. For fluorescencemeasurements the pH of the serum was adjusted to about pH 9.

Except for the incident wavelength, the same method of light detectionwas used for all samples in 6 mm by 50 mm glass tubes. The serum isdescribed in Example 7.

Measurements were made in the stated concentration of serum. The lightsignal value of a tube containing only serum at the proper concentrationwas subtracted from the gold particle or fluorescein value in order toobtain the light signal due to just the fluorescein or gold particles.The following steps were performed for the measurement of the lightsignal from particles.

A. (a) Determine the light signal emitted from a control tube containingserum at the proper concentration but no particles.

(b) Determine the light signal emitted from a tube containing particlesat known concentration.

(c) Subtract the control light signal value (a) from the light signalvalue of (b).

The following steps were performed for the measurement of the lightsignal from the fluorescent solution.

B. (a) Determine the light signal emitted from the control tube.

(b) Determine the light signal emitted from a known concentration offluorescein in a tube.

(c) Subtract the control light signal value (a) from the light signalvalue of (b).

C. (a) Compare light signals obtained from known concentrations ofparticles.

Example 9

Detection Limits for Polystyrene, Polystyrene-fluorescent compound, andGold Particles at High Serum Concentration

The results are given in Table 19. Measurements were made in the statedconcentration of serum. The light signal value of a tube containing onlyserum at the proper concentration was subtracted from the gold particleor polystyrene particle value in order to obtain the light signal due tojust the polystyrene or gold particles. No optical filtration was done.

The following steps were performed for the measurement of the lightsignal from particles.

A. (a) Determine the light signal emitted from a control tube containingserum at the proper concentration but no particles.

(b) Determine the light signal emitted from a tube containing particlesat known concentration.

(c) Subtract the control light signal value (a) from the light signalvalue of (b).

(d) Compare light signals from known concentrations of particles.

Except for the incident wavelength, the same method of light detectionwas used for all samples in 6 mm by 50 mm glass tubes. The serum isdescribed in Example 7.

Example 10

At Low concentrations of Gold Particles Serum has no effect on LightScattering Properties of the Gold Particles

The results are presented in Table 20. The serum at 95.7 percentconcentration is clear and straw colored and has an absorbance of 0.14at one cm pathlength and incident light wavelength of 543 nm. The lightscattering measurements were made in 6 mm by 50 mm glass tubes with aninner diameter of about 5 mm. On the basis of the difference in theabsorption of both incident and scattered light of a wavelength of 543nm the light scattering signal from gold particles present in the serumsample should be roughly 80 percent of the signal from the sameconcentration of gold particles present in water. No optical filters areused in this example.

The following steps were performed.

(a) Illuminate all samples with the same incident light composition andintensity.

(b) Determine the light signal emitted from a control tube containingwater or a proper concentration of serum but no particles.

(c) Determine the light signal emitted from a tube containing particlesat a known concentration.

(d) Subtract the control light signal value (b) from the light signalvalue of (c).

(e) Compare light signals from equal concentrations of gold serum andwater.

Example 11

Preparation of a 16 nm Gold Particle Suspension

2.5 ml of sterile water was added to 0.1 g HAuCl₄.3H₂O to form a 4%HAuCl₄.3H₂O solution. The solution was centrifuged to remove particulatematter. In a separate flask, 10 ml of sterile water was added to 0.1 g.of sodium citrate to form a 1% sodium citrate solution. The citratesolution was filtered through a 0.4μ polycarbonate membrane filter toremove particulate matter. To a very clean 250 ml Erlenmeyer flask, 100ml of sterile water and 0.25 ml of the 4% HAuCl₄.3H₂O was added. Theflask was placed on a stir hot plate at a setting of 4 and covered witha 100 ml beaker. When the mixture started boiling, 2 ml of the 1% sodiumcitrate was added. The solution turned a black color within a minuteafter adding the citrate. It then turned purple and finally a deep red.The red color was achieved after about 2 minutes after adding thecitrate solution. The mixture solution was boiled for 30 more minutesand then cooled to room temperature and sterile water was added to bringthe total volume to 100 ml. The final gold concentration is about 0.005%and particle concentration is 1.2×10¹² particles/ml, assuming that allthe gold salt was converted to gold particles.

Example 12

Stabilization of Metal Particles with Polyethylene Compound

1 gram of the PEG compound (MW 20,000) was added to 100 ml of sterilewater to form a 1% PEG compound solution and the solution was filteredthrough a 0.4μ polycarbonate filter using a 50 ml syringe. To stabilizea given volume of particles, add the volume of particle solution to avolume of 1% PEG compound solution that gives a final PEG concentrationof 0.1%.

Example 13

Preparation of 30 nm Silver Coated Particles from 5 nm Diameter GoldParticles

10 ml of sterile water was brought to a boil in a 30 ml beaker. 2 mg ofgelatin was then added slowly and the solution was allowed to continueto boil with stirring until all of the gelatin was dissolved. Thesolution was then cooled to room temperature. 2 ml of a 47% citratebuffer pH 5 was added. 0.18 ml of a solution containing 5 nm goldparticles (at a concentration of about 0.005% gold, 3.8×10¹³ goldparticles/ml) was added followed by the addition of 3 ml of a 5.7%hydroquinone solution. The mixture was mixed well, followed by additionof sterile water for a final volume of 20 ml. 50 μl of a 4% silverlactate solution was added in 10 μl increments and the mixture wasstirred rapidly by hand. The final silver concentration is about 0.005%and the final silver coated particle concentration was about 3.4×10¹¹particles/ml. Assuming that all of the added silver had depositedequally on each gold particle, the particle size was calculated to be 30nm. After the final addition, the sol appeared bright yellow in roomlights. In bulk solution, the light scattered by a diluted volume of thesol contained in a 6×50 mm glass tube was blue when illuminated by anarrow beam of white light. When a dilution of the silver sol wasexamined microscopically with the SpectraMetrix microscope under DLASLPDconditions with a 10× objective and 12.5 eyepiece, a mixture of brightparticles with different colors could easily be seen. The particlesdominant in number were purple-blue particles. Yellow, green and redparticles were also present. By adjusting the concentration of the 5 nmdiameter gold particles that we use in the procedure described here, wemade silver coated particles with diameters in the range 20 to 100 nm.

Example 14

Scattered Light Properties of Nonspherical Silver Particles Formed andExamined on a Microscope Glass Slide

A small drop of a diluted, silver particle sol prepared as described inexample 13 was placed on a microscope glass slide and covered with acover glass to form a very thin film of sol between the cover glass andmicroscope slide. When a spot of the thin silver sol film wasilluminated by a very narrow beam of light and viewed by the naked eye,at an angle which prevented the incident light from entering the eye,the illuminated spot had a blue scattered light appearance. The silversol film was then examined microscopically with a light microscope underDLASLPD conditions with 10× objective and 12.5× eyepiece. It wasobserved that in a few minutes most of the particles became attached toand immobilized on the surface of the glass slide and cover glass. Bluecolored particles were the most abundant. We then discovered that when apoint on the cover glass was pressed with the point of a fine needleprobe, the particles in the pressed area permanently changed color fromtheir original blue (scattered light detection). At the center of thepressed area the particles were red. This center spot was surrounded byconcentric circles of different colors. From the center on out, thecolors changed from red, to green to yellow to blue. The red, green andyellow particles were very bright. Theoretical calculations which wehave done indicate that small silver particles have a blue color. Theeffect of pressing seems to be to change the shape of the particles. Ourresults therefore show that small silver particles can be converted fromtheir original blue scattered light color to other colors of scatteredlight by changing their shapes. By moving the cover glass we found thatwe could disperse the differently colored particles in the pressed areainto the liquid phase. In this phase the particles underwent Brownianmotion and the light scattered by green and red particles flickered,which is expected for nonspherical particles.

Example 15

Preparation of Larger Diameter Gold Particles from 16 nm DiameterParticles

A 2.4% solution of hydroxylamine hydrochloride was made by adding 24 mg.of hydroxylamine hydrochloride to 1 ml of sterile water, mixing and thenfiltering through a 4μ polycarbonate membrane filter attached to a 10 mlsyringe. A solution of 4% HAuCl₄.3H₂O was made by adding 2.5 ml ofsterile water to 0.1 g HAuCl₄.3H₂O in a test tube mixing and thencentrifuging to remove particulate matter. 25 ml of sterile water wasadded to a 250 ml Erlenmeyer flask, followed by addition of the volumeof 16 nm gold particles shown in Table 1 depending on the desiredparticle size. Next we added the volume of the 4% HAuCl₄.3H₂O solutionspecified in Table 1. Finally we added sterile water to bring the totalvolume to 100 ml. Then the volume of the hydroxylamine hydrochloridesolution specified in Table 1 was added with rapid hand stirring and themixture was allowed to sit for 30 minutes. Within seconds after addingthe hydroxylamine hydrochloride solution, the solution turned from aclear, slightly pink color to a final clear red or murky brown color,depending on particle size. The smaller sizes give red coloredsolutions.

TABLE 1 Desired Au Particle 16 nm Gold HAuCL₄.3H₂O Hydroxyl-amineDiameter, nm Sol, ml solution, ml solution ml 40 6.4 0.234 1.25 60 1.90.245 1.25 80 0.8 0.248 1.25 100 0.41 0.249 1.25

Larger diameter particles were prepared following the same procedure asdescribed above, but using the specified volumes of solutions asdescribed in Table 2 and using the 100 nm diameter Au particle solutioninstead of the 16 nm gold solution.

TABLE 2 4% Hydroxyl HAuCl₄.3H₂O amine Desired Au Particle 16 nm GoldSolution, Solution Diameter, nm Sol, ml ml ml 200 12.5 0.219 1.25 4001.56 0.246 1.25 600 0.436 0.249 1.25 800 0.195 0.25 1.25 1000 0.1 0.251.25 2000 0.012 0.25 1.25

Example 16

Preparation of a Silver Coated Particle from 16 nm Gold Particles

25 ml of sterile water was added to a 250 ml Erlenmeyer flask followedby the addition of 6.4 ml of a 0.005% 16 nm gold particle sol and theresulting solution was mixed. 0.234 ml of a 40 mg/ml L(+) Lactic Acidsilver salt solution was then added. A deep purple color was immediatelyseen. Enough sterile water was then added to bring the total volume to100 ml. While rapidly stirring by hand, 1.25 ml of a 24 mg/ml solutionof Hydroxylamine Hydrochloride was added and the resulting sol appearedlavender silver in color. A small drop of the sol was placed on a glassslide, covered with a cover glass and examined with a light microscopeunder DLASLPD conditions. Red, green, purple, and yellow particles wereseen. The scattered light color of a dilute solution of these particlesin a test tube with white light illumination was ice blue.

Example 17

Preparing BSA Coated Glass Slides

A model system was setup to study the signal and detection parametersfor various combinations of particles and illumination and detectionmethods for detecting particles on a solid phase as in a solid phaseassay.

This system involved first coating glass slides with bovine serumalbumin (BSA) and then depositing different amounts of gold particles inspecified areas to study the parameters. Here we discuss the methodwhich we use to coat the slides with BSA.

A 10% BSA in water solution was made by adding 1.5 g BSA to 15 ml ofsterile ultrapure water mixing and then filtering the solution with a0.44 mm polycarbonate membrane. A 0.02% BSA (200 μg BSA/ml) solution wasmade by adding 20 μl of the 10% BSA solution to 10 ml of sterile waterand filtering the BSA solution through a 0.4 mm polycarbonate membrane.

Ordinary microscope glass slides were cleaned by scrubbing with a brushdipped in methanol. After brushing, the slide was then cleaned bysquirting the slide with sterile water using a plastic squirt bottle. Aglass slide was coated with BSA by submerging the slide in a beakercontaining 0.02% BSA in sterile water and incubating for 1 hour. Theslide was then removed from the beaker and rinsed by squirting sterilewater from a squirt bottle. Both sides of the slide were rinsed. Theslide was then submerged in 150 ml beaker full of sterile water forabout 10 min. It was rinsed again by squirting water. It is mostimportant to remove free BSA from the slide because free BSA hinders thebinding of metal particles to the coated slide. The BSA coated glassslide is then dried and stored dry in a clean plastic box.

Example 18

Depositing Gold Particles on a BSA Slide

Small circles (about 8 mm diameter) were scribed on the BSA coated glassslides using a diamond scriber to mark the areas where gold particleswere to be deposited. 3 μl of an unprotected gold particle solution withthe desired gold particle concentration is deposited on one of themarked areas of the slide. The gold particle solution is deposited onthe opposite side of the actual scribe marks to prevent interaction ofthe gold particle solution with the scribe marks.

To prepare a series of patches of gold particles, on a glass slide, inwhich the gold particle density is systematically decreasing, it isdesirable to have the series of patches on a line in the center of theslide. To achieve this alignment we mount the slide on a holder, whichwe made, which allows us to deposit the gold patches in the correctalignment. It should be noted that the patches cannot be seen in roomlights (that is, the particle density is so low that they have no colorin room lights). We thus make a mark on the side of the slide toidentify the position of the patches. The marks are made as we depositthe particles. To form a patch of particles, we deposit 3 μl of theunprotected gold solution diluted to the desired gold particleconcentration. The slide is then incubated for a specified time in aclosed plastic box. The interior walls and bottom of the box are coveredwith a wet paper towel to prevent evaporation of the gold sol on theslide. The slide is then removed and rinsed by squirting sterile waterwith a pasteur pipette. We have found that for the most efficientbinding of gold particles to the BSA on the slide, the pH of the goldparticle solution should be adjusted to the pI of BSA (pI=4.58-5.45).

Example 19

Microarray Analytical Assay—Binding 60 nm Diameter Gold Particles Coatedwith BSA—Biotin to Discreet Individual 80 Micron Diameter StreptavidinPatches on a Plastic Substrate

The following solutions were prepared. A 1 mg/ml BSA-Biotin solution wasmade by adding 2 mg of BSA-Biotin to 2 ml of sterile water and dialyzingagainst distilled water in a 500 ml Erlenmeyer flask for several hoursat room temperature. The water was changed 4 times. The last waterchange was sterile water. A 20 mM Tris-Saline, 0.1% PEG Compound, 0.02%Na Azide pH 7.4 buffer was also prepared. All solutions were filteredthrough a 0.4μ polycarbonate membrane filter. Polystyrene test tubeswere washed very well with sterile water using a squirt bottle andfilled with 4 ml of the 60 nm diameter gold particle solution andcentrifuged in the clinical centrifuge for half an hour. The particleswere then washed as described elsewhere. The soft pellets wereresuspended in 10 ml of sterile water. The pH of the gold particlesolution was adjusted as follows: 100 μl of 1% PEG compound solution wasadded to a clean polycarbonate test tube. To this 1 ml of the 60 nm goldsol was added and incubated for 2 minutes. 0.02M K₂CO₃ was added to thegold sol in increments of 2 μl until pH 6.6 was achieved. The number ofμl of 0.02M K₂CO₃ needed to adjust the pH is then calculated to add tothe remaining ml of the gold sol, in this case it was 80 μl. 9.5 ml ofthe pH 6.6 gold sol was then added to 1.15 ml of a 1 mg/ml BSA-Biotinsolution in a polycarbonate tube, and incubated for 5 minutes at roomtemperature. The solution was then centrifuged for half an hour in theclinical centrifuge and the supernate was then decanted. The remainingsoft pellet was resuspended in 3 ml of sterile water and thencentrifuged as previously described, supernate decanted, and thenresuspended in 0.1% PEG compound solution and centrifuged again. Thesupernate was decanted and the pellet was resuspended in 20 mMTris-Saline, 0.1% PEG compound solution and centrifuged The supernatewas then decanted leaving behind about a 200 μl soft pellet. 50 μl ofthis solution was added to the plastic wells that contained the 80μdiameter streptavidin spots and the wells were incubated overnight in ahumid chamber. The wells were then washed several times with sterilewater using a pasteur pipette to squirt and remove water from the well.For detection with the microscope the wells were filled with 60 μl ofsterile water.

Example 20

Detection of Bound 60 nm diameter Gold Particles Coated with BSA-biotinto a Microarray of 80 Micron Diameter Streptavidin Coated Binding SiteSpots

A suspension of the BSA-biotin-Au binding agent(60 nm diameter goldparticle) was added to plastic wells which contain the microarray ofstreptavidin 80μ diameter spots on the bottom surface of the well. Aftera suitable incubation time, the wells were washed and viewed with ourdeveloped light microscope system under DLASLPD conditions. We observedthe BSA-biotin-Au labeled particles bound to the individual 80uindividual spots. The 80μ streptavidin spots which were not visibleprior to the addition of the particles, appeared as bright fairlycircular spots. Individual spots containing different BSA-biotin-Auparticle surface densities were obtained by incubating for differenttimes or by using different BSA-biotin-Au concentrations. We were easilyable to detect by eye with our microscope at magnification of about200×, individual particles bound to the streptavidin spots at lowbinding densities. To automate the counting and integrated lightintensity measurements from individual 80u spots, we tested video imageprocessing software which we had on 24 hour loan from a Company here inSan Diego. We captured video images using an inexpensive black and whitevideo camera, a video frame grabber, and a simple desktop computerimages which contained the 25 individual spots that were in the arraydevice well. The software was able to measure the integrated lightintensity of each spot as well as the number of particles per individualspot. For example, a streptavidin spot that was labeled with a lowdensity of BSA-biotin-Au binding agent was analyzed with the videoimaging system using a particle counting mode. To get some idea of thesignal to background, an 80u diameter spot of solid-phase area notcoated with streptavidin was analyzed to determine the background. Inthis preliminary model system, the signal/background was 317/25˜13 witha labeling density on the spot of about 0.06 particles/u² undernon-optimized illumination and detection conditions. Based on thisnon-optimized preliminary data, these data imply that at asignal/background of 3/1 particle densities of 0.015 particles/u² aredetectable. Under more optimized conditions, the lower level ofsensitivity may be much lower.

Example 21

Detection Sensitivity of Gold Particles in Thin Films

60 nm gold sol was diluted by factors of 10 and 20 μl of each dilutionwas deposited as spots on a BSA coated slide. The slide, with no coverglass was then placed on a Porro prism with immersion oil. Each gold solspot had a diameter of about 4 mm. The following table gives pertinentinformation on each spot.

SPOT Particles/ Diameter Au Sol M ml (mm) # of Particles Observation* 3× 10⁻¹¹   2.3 × 10¹⁰ 12.4 4.6 × 10⁸ Very Intense Yellow 3.8 × 10⁻¹²  2.3 × 10⁹ 12.8 4.6 × 10⁷ Intense Yellow Green 3.8 × 10⁻¹³ M 2.3 × 10⁸11.4 4.6 × 10⁶ Weak but detectable green *As detected by eye

The table below expresses the above data in units that are moremeaningful in clinical assay applications. The height of the liquid inthe spot can be calculated with the expression

h=V/A

where V is the volume of liquid in the spot (20 ml=0.02 cm³) and A isthe area (in cm²) of the spot. Using A=1.2 cm² we get h=0.016 cm=160 m.This height is much smaller than the depth of field of the eye or ourelectronic and optical methods of detection and thus each spot behaves(from a geometrical point of view) as if all of the particles were onthe surface of the slide and the sensitivity reported in the table aresimilar to the sensitivities expected for particle deposited on thesurface.

Spot Spot Area, Area, Particles ^(#)Particles *Intensity Au M cm² μ² perSpot per μ² of Spot 3.8 × 1.2 1.2 × 4.6 × 10⁸ 3.83 Very 10⁻¹¹ 10⁸Intense, Yellow 3.8 × 1.3 1.3 × 4.6 × 10⁷ 0.35 Intense 10⁻¹² 10⁸ YellowGreen 3.8 × 1.2 1.2 × 4.6 × 10⁶ 0.038 Weak but 10⁻¹¹ 10⁸ Detectablegreen *As detected by eye ^(#)This column was calculated by dividing thenumber of particles per spot by the area of the spot.

Example 22

60 nm Gold Particles Deposited on the Surface of a BSA Coated GlassSlide

A series of 60 nm gold particle solution dilutions were formed and 3 μlof each dilution was deposited as a small spot on a BSA coated slide.The spots were in a row on the same slide. The slide was incubated in ahumid chamber for 6.5 hours, then rinsed with sterile water. Theparticle density on each spot was determined by particle counting underDLASLPD conditions in a light microscope which has an eyepiece with acalibrated reticle. The following table shows our results.

Deposit Total number of ^(#)Particles Area concentration* particles per100 Number particles/ml deposited micron² 1  2.3 × 10¹⁰ 2.3 × 10⁸ 460 22.1 × 10⁹ 2.1 × 10⁷ 41.8 3   7 × 10⁸   7 × 10⁶ 13.9 4 3.5 × 10⁸ 3.5 ×10⁶ 7 5 1.75 × 10⁸  1.75 × 10⁶  3.48 6 8.75 × 10⁷  8.75 × 10⁵  1.74*Deposit concentration is the concentration of unprotected gold solsolution that was placed on top on a spots. ^(#)Particles per micron² -This gives the number of particles per 100 micron square if all of theparticles in the solution deposited on a specified are became attachedto the slide. The area covered by the solution has a diameter of about 8mm. The area is A = 3.1416*(.4)² cm² = 0.5 cm² = 0.5 × 10⁸ micron².

After 6.5 hours, the slide was washed by gently squirting sterile wateron each gold containing area on the slide. The slide was dried with heatgun on cold setting. The dried slide was viewed under DLASLPD conditionswith a light microscope and the particle density in each area wasdetermined using the calibrated reticle in the microscope eyepiece tocount particles/ reticle square. The following table shows the results.

Particles Particles counted Particles Sam- deposited/ using reticlecounted per 100 ple Eye* 100 micron² (area, micron²)# micron² 1 Veryintense 460 20** (39)  51.2 yellow green 2 Intense 41.8 7 (39) 18 yellowgreen 3 Fairly 13.9 13 (100) 13 intense yellow green 4 No intensity 7 00 detected 5 No intensity 3.48 0 0 detected 6 No intensity 1.74 0 0detected *Eye - This is the light intensity excited by the microscopeilluminator under DLASLDP conditions and viewed by eye from the gold onthe specified area of the slide. **The particles in this area were toonumerous to count and the count listed here may be in error by as mushas 2×. # - The area listed in parenthesis is the area of 1 reticulesquare for the objective and optovar setting used to count particles.Particles counted per Sample micron² Eye* 1 0.512 Very intense yellowgreen 2 0.18 Intense yellow green 3 0.13 Fairly intense yellow green 4 0No intensity detected 5 0 No intensity detected 6 0 No intensitydetected

Example 23

Sensitivity for Visual Detection of Intensity from a Liquid Spot of 60nm Gold Particle

Two dilutions of 3.4×10⁻¹² M (0.005% gold) 60 nm gold sols were preparedand 2 mL of each dilution was deposited in a separate spot on a glassslide. Each spot had a diameter of about 4 mm (Area=6.28×10⁶ m²). Thedifferent spots were in a row in the middle of the same slide. Particleconcentrations and densities in each spot are shown in the followingtable.

Gold Sol M Particles/ml Particles/μl Particles/μ²  3.4 × 10⁻¹² M  2.1 ×10⁹  2.1 × 10⁶ 0.31 1.05 × 10⁻¹² M 1.05 × 10⁹ 1.05 × 10⁶ 0.155  0.5 ×10⁻¹² M  0.5 × 10⁹  0.5 × 10⁶ 0.077 0.25 × 10⁻¹² M 0.25 × 10⁹ 0.25 × 10⁶0.0385

To determine the lowest particle density from scattered light intensityas detected by the unaided eye, we placed the slide on a Porro prism bymeans of immersion oil. Each spot, still in liquid form, wassequentially illuminated by light from the Baush-Lomb Illuminator with a×10 objective at the end of the fiber. The spot produced by theilluminator was about 4 mm in diameter. In the dark room, at night, wecould see down to 0.0385 although the latter could just barely be seen.

Example 24

Sensitivity for Photodiode Detection of 60 nm Gold Particles (insuspension) at Different Concentrations in Immulon Plastic microtitierWells

Different dilutions of 60 nm gold sol were placed in different ImmulonWells (200 μl in each well). To measure scattered light intensity, thebottom of a well was illuminated with white light from a LeicaMicroscope Illuminator that was equipped with ×10 objective. The bottomof the well was a few mm from the objective. The light from theobjective produced a beam that was focused on the center of the well.The beam diameter at the focal point was about 5 mm. Scattered light wasdetected by a photodiode that was positioned to detect light through theside wall of the well (right angle detection). The scattered light wasdetected through a small hole (diameter about 1 mm) that was positionedin front of the photodiode to limit background light detection. Thewells containing the different gold sol dilutions were attached to eachother and each well can be sequential positioned in the illumination anddetection paths. The output of the photodiode is measured with anoperational amplifier that is operated in the current mode. The feedbackresistor of the op amp determines the sensitivity of the amplifier. Thephotodiode is operated in the photovoltaic mode. Two sets of 60 nm golddilutions were prepared and the intensities measured with thephotodiode.

a. First set of dilutions

The master solution (3.8×10⁻¹¹ M) was diluted by factors of two. Thefollowing readings were obtained. Readings were made with a 5 megohmresistor in the feedback loop of the op amp.

Gold Particle Concentration Intensity (Volts)  1.9 × 10⁻¹¹ M 3.27 0.95 ×10⁻¹¹ M 1.6 4.75 × 10⁻¹² M 0.89 2.38 × 10⁻¹² M 0.44  1.2 × 10⁻¹² M 0.240 0.075

b. Second dilution

A ×11 dilution solution (3.4×10⁻¹² M) was diluted by factors of ×2.Results are as follows.

Gold Particle Concentration Intensity (Volts)  3.4 × 10⁻¹² M 0.614  1.7× 10⁻¹² M 0.378  8.5 × 10⁻¹³ M 0.198 4.25 × 10⁻¹³ M 0.147 2.13 × 10⁻¹³ M0.100 1.06 × 10⁻¹³ M 0.086 0 0.075

The above results show that, in wells, we can detect 60 nm diameter goldparticles in the range 1.9×10⁻¹¹ M to 1×10⁻¹³ M. The upper range can beextended.

Example 25

Reproducibility for Depositing and Visually Detecting (IntegratedScattered Light Intensity) 60 nm Gold Particles Deposited on a BSACoated Glass Slide

3 μl of a 2× dilution of 0.005% gold (60 nm) solution was deposited ineach of 5 spots on a BSA coated slide. The slide was incubated for fiveminutes and then introduced into a beaker containing 150 ml of distilledwater. The water washed the unbound gold off the slide. The spots werethen illuminated with our illuminator (white light SpectraMetrixIlluminator). The gold particles in each spot were in the form of a ring(that is the particles were not homogeneously distributed in the spotbut were confined to a ring) which scattered green light and could beclearly seen in the dark room with the unaided eye.

The experiment was repeated with a newly coated BSA slide except thatduring the incubation of the gold dots, the slide was gently tapped onthe side with the finger so as to stir the liquid in the gold particlespots. After 5 minutes, the slide was introduced into 150 ml of sterilewater in a beaker and the light scattered by each spot was viewedalternatively using the white light SpectraMetrix Illuminator. Theilluminator produced a spot of light of about 5 mm diameter on theslide. Gold spots could clearly be seen through scattered light wherethe gold sol was deposited. The spots were viewed with the slidesubmerged in water which reduced scattering by imperfection on theslide. All of the spots scattered green light and had about the sameintensity as evaluated by visual detection. A small, non-lightscattering spot (dark spot) appeared in the center of each spot.

Example 26

Color of Light Scattered by 60 nm Gold Sols at Different Gold ParticleConcentrations

Six 8×50 mm (1.6 mL) polystyrene tubes were washed by rinsing withsterile from a squirt bottle. Excess water was removed from each tube byshaking but the tubes were not dried. A gold particle solution (60 nm,0.005%) was then serially diluted by factors of 1,2,4,8,16 and 32. Eachtube had 500 mL of gold particle solution. The diluted gold sol wasstable (scattered light did not change color on standing) in thepolystyrene tubes. No evidence of aggregation. The light scattered bythe different dilutions had the following colors. The gold sol used inthese observations had been washed several times with sterile water toremove salts (that are used to form the gold sol) which seem todestabilize the gold particles.

Dilution Color 1 yellow green 2 yellow green 4 light green 8 light green16 light green 32 light green

Example 27

Stabilization of Gold Particles with BSA

We found that 900 mg of BSA were required per ml of 60 nm, 0.005% goldsol to stabilize the gold sol against agglutination by 1% NaCl.

Example 28

Deposition of 60 nm Gold Particles at High Particle Densities on BSACoated Glass Slides

This example shows how to deposit spots of different surface densities(25 to 100 particles/μ²) of gold particles on a glass slide. These spotsare used in examples 30 and 31 to determine the intensity, color andhomogeneity of light scattered from these spots (white lightillumination) as seen by the naked eye and in a light microscope usingDLASLPD methods.

4 ml of 60 nm gold sol (0.005%, 3×10¹⁰ particles per ml) werecentrifuged in the clinical centrifuge at maximum speed until all of thegold particles sedimented to the bottom of the tube (about 30 min). Thesupernatant was removed and the soft pellet was diluted by factors of 1,2 and 4. We estimate that the soft pellet had a particle concentrationof about 3×10¹¹ particles per ml. 4 ml of each dilution was deposited onseparate spots on a BSA coated glass slide and the liquid in each spotwas allowed to evaporate at room Temperature. The number of particlesdeposited in each spot (assuming that the ×1 gold sol had aconcentration of 3×10¹¹ particles per ml) is shown in the followingtable. It should be noted that the maximum particle density which can beachieved with 60 nm particles (saturated monolayer of particles) is 354particles/μ².

Particles Dilution Deposited^(#) Particles/μ²* 1 1.2 × 10⁹   100 2 6 ×10⁸ 50 4 3 × 10⁸ 25 *Particle Density in particles/μ² is calculated fora spot with a diameter of 4 mm (4 × 10³μ) and area of 12.6 × 10⁶ μ².^(#)Calculated for a deposit of 4 μl of a 3 × 10¹¹ particles per ml 60nm gold sol.

After solvent evaporation, each spot was examined for its appearance inroom lights and under DLASLPD illumination (as viewed with unaided eye).DLASLPD illumination was with a Leica microscope illuminator that had a×10 objective focusing lens and produced a narrow beam of light thatfocused to a small spot at a distance of about 10 mm from the objective.The spots were also examined by light microscopy using DLASLPD methodswith ×2.5, ×10, ×25 and ×40 objectives and plus additional magnificationof ×1.25, ×1.6, and ×2. Only a small area of each spot could be seen ata time with the ×10 and ×20 objectives. However, the whole spot could beseen with the ×2.5 objective. To determine the particle surface densityon each spot, we counted the particles seen through the microscope on agiven area and divided by this number by the area. The area wasdetermined with a reticle positioned in the ocular of the microscopewhich had been calibrated with slide micrometer. As an example, whenparticle counting was done with the ×40 objective and additionalmagnification of 1.25 and 2 (before the ocular), the unit square in theocular reticle used for particle counting were respectively 6.25μ×6.25μ(area of square=39.1μ²) and 10μ×10μ (area=100μ²). These are the areas inthe object plane.

Example 29

Observations of High Surface Density Gold Particle Spots in Air

In this example, the gold particle spots prepared as described inexample 29 are examined visually and microscopically using DLASLPDillumination. The spots were dry and the gold particles were thus inair.

a. ×1 dilution spot

In room lights the spot has a dark purple appearance with a lighter spotof less than 1 mm diameter in the center. Under DLASLPD illumination thespot had a rather uniform whitish orange appearance. The spot was highlyintense. Under DLASLPD conditions in the microscope (×10 objective,extra magnification ×2, 12.5 ocular) the spot as viewed through theocular had a highly intense orange color. Individual particles couldeasily be seen. Some particles could be seen very close to each other oreven overlapping. The majority or particles are orange but some aregreen. Two or more particles which are separated from each other by lessthan about the spatial resolution of the microscope appear as singleparticles. If the space between the particles are close enough toperturb their light scattering properties, the particle group appears asa single particle of a color that is different than that of the singleparticle. At high particle density, it is expected from theoreticalcalculations that many particles will be separated by distances whichare smaller than the resolution of the microscope. The appearance of thespot did not change much when viewed with a ×10 or ×20 objective. Thearea on the slide which is outside of the spot (background) was verydark compared to the intense spot. With the ×2.5 objective, the wholespot could be seen. It had an intense orange appearance with a smallgreen ring at the periphery. The color seemed to be fairly uniform inthe orange area although same patches seemed to be lighter than others.The particle surface density in the green ring was much lower than inthe orange area.

b. ×2 dilution spot

In room lights, the spot has a medium purple outer ring with a darkpurple spot of about 2 mm in the center. Under DLASLPD illumination thespot had a rather uniform whitish yellow appearance. The spot was highlyintense. Under DLASLPD conditions in the light microscope (×10 and ×20objectives, ×2 extra magnification, 12.5 ocular) the spot as seenthrough the ocular had a highly intense orange color but the color wasnot as uniform as ×1 dilution. There are patches which have a greenishappearance. Very closely spaced particles could be seen. The majority ofthe particles are orange but there are some green which are in highabundance in the green patches. The appearance of the spot did not verymuch when viewed with a ×10 or ×20 objective. The area outside of thespot was very dark compared to the intense spot. With the 2.5 objectivethe whole spot could be seen. It had an intense orange appearance with asmall green ring at the periphery. The color seemed to be fairly uniformin the orange area although same patches seemed to be lighter thanothers. Some of this non-uniformity is due to inhomogeneities in theilluminating system which had not been optimized.

c. ×4 dilution spot

In room lights, the spot had a very light purple color with a small darkspot (about 1 mm diameter) displaced to one side of the circle. UnderDLASLPD illumination the spot had a rather uniform whitish greenappearance. The spot was highly intense. Under DLASLPD conditions in thelight microscope (×10 objective, ×2 extra magnification, 12.5 ocular)the spot appeared to be highly non-uniform probably to unevenevaporation of solvent. The center of the spot had a highly intenseorange or lavender color. Particles are very close to each other or evenoverlapping in this central area, with most particles having an orangecolor and some a green color. Away from the center, the spot had agreenish appearance with a predominance of green particles with someorange particles. There are alternating rings of green and yellow coloras one goes from the center of the spot to the periphery. The areaoutside of the spot (background) was very dark compared to the intensespot. The whole spot can be seen with ×2.5 objective. The spot had anoval appearance with a orange or lavender spot (about 1.5 mm diameter)towards the center. This was surrounded by intense circles ofalternating yellow green and green area. A small ring at the peripheryhad a less intense (but still intense) color which was distinctly green.In this peripheral area, almost all of the particles are green particlesand could be counted with ×40 objective, extra magnification ×2. Theparticle surface density in the green particle area was about 20particles/39.1μ² or 0.5 particles per μ². At the very periphery theparticle count dropped rapidly and we counted about 7 particles per100μ² or 0.07 particles/μ². The gradient of particles on this spotallows us to count the particles up to the counting limit of about 1particle/μ².

Conclusions for immobilized particles in air

a. The procedure described above allows us to deposit gold particles athigh surface densities on small (4 mm) spots. The deposits are notcompletely uniform when viewed in room lights as expected for theevaporation process which we use to form the spots.

b. Under DLASLPD conditions in the light microscope, the ×1 and ×2dilution spots are fairly uniform. For the ×4 dilution, the spotdisplayed more non-uniformity.

c. The particle density of the spots seems too high for particlecounting to be meaningful (particles to close to be resolved asindividual particles). However, at the periphery of the ×4 dilution spotthe particles could be counted and the density here was around 0.5particles/μ². This particle density is close to the maximum particledensity which can be counted with the resolution of our microscope.

Example 30

Observations with High Surface Density Gold Particle Spots in Water

The slide used in example 30 was submerged in 150 ml of sterile water ina beaker. The particles did not seem to come off the slide. Using themicroscope illuminator with 10× objective, the gold spots in thesubmerged slide could each be separately illuminated with a narrow beamof light. The color of the spots (observation with unaided eye) did notseem to change in going from air to water. The glass slide was removedfrom the beaker of water and covered with a cover glass. A thin film ofwater surrounded the gold particles. Microscope observations were asfollows.

a. ×1 dilution spot

When viewed under DLASLPD conditions with a light microscope with ×2.5objective, ×1.25 extra magnification and ×12.5 ocular, the spot had afairly uniform orange lavender appearance. The periphery of the spotcontained bright, yellow green particles. The particles at the peripherycan be easily seen with 10× to 40× objectives. The particle surfacedensity was very high throughout the spot even at the periphery exceptat a very thin ring at the very periphery where the individual particlescan easily be seen as bright objects.

b. ×2 dilution spot

The whole spot could be seen through the 12.5× ocular using the ×2.5objective and ×1.25 extra magnification. The spot had a very intenseyellow color overall and seemed fairly uniform. Most of the spot wasyellow but towards the periphery the spot had a yellow green color. Witha ×40 objective and ×1.25 extra magnification, the particles at a veryhigh surface density could be seen. Most of the particles have a yellowgreen color. A few have a green or red color. The spot had a very fairlyuniform intensity except at the periphery where the particle densitydrops off very rapidly to zero (dark background). The individualparticles with dark spaces between then can easily be seen and countedat the periphery where the particle surface density is low.

c. ×4 dilution spot

The whole spot could be seen with the 2.5× objective plus 1.25× extramagnification. The spot had a very intense yellow green color and thecolor was very uniform in contrast to the observations in air where thespot had many green patches. Individual particles could easily be seenwith ×40 objective and ×1.25 extra magnification. The particles in waterare much more intense or brilliant than in air. The particles at theperiphery were not predominantly green but in water most of theparticles were yellow green with some red and orange particles. Most ofthe spot had a very intense yellow color. Individual particles can beseen with ×40 objective but the particles are very dense and overlap. Inthe most intense area of the spot, the particles seen with ×40 objectiveand ×2 extra magnification, were at a density of about 25particles/39.1μ² or about 0.6 particle/μ². This number may not representthe true particle surface density because of limitations microscoperesolution.

Conclusions for immobilized particles covered by water

Placing the gold spots in water seems to give them a more uniformappearance. Spots ×2 and ×4 dilutions seem to both have a yellow colorwhen viewed by the unaided eye with the illumination of the lightmicroscope under DLASLPD conditions (slide sitting on prism and coupledto the prism with immersion oil). The ×1 dilution spot appears to beorange to the eye.

Example 31

Binding of 60 nm Gold-BSA-biotin Reagent to Magnetic Beads

This example demonstrates our ability to detect and quantify thespecific binding of gold particles to magnetic beads by light scatteringintensity measurements in suspension and to detect individual goldparticles bound to magnetic beads by light microscopy under DLASLPDconditions.

60 nm gold particles were coated with BSA that had been covalantlylabeled with biotin (BSA-biotin-Au). 500 μl of a phosphate bufferedsaline solution, pH 7.4, containing 0.1% BSA solution was added to eachof 5 microcentrifuge tubes. The tubes were labeled 0, 1, 2, 3, 4. Asolution of BSA-biotin-Au at a gold particle concentration of 3.8×10⁻¹¹M was added to each tube and the tubes were shaken. Additional amountsof the BSA-biotin-Au solution were added to bring the concentration ofgold BSA-biotin particles=8×10⁻¹³ M in each tube. The following amountsin μl of Dyna beads M280 Streptavidin (2.8μ diameter beads withstreptavidin covalantly attached to the surface of the bead) suspensioncontaining 6.7×10⁸ Dyna beads/ml (10 mg/ml) or about 1×10⁻¹² M beadmolar concentration, dissolved in phosphate buffered saline (PBS), pH7.4, containing 0.1% BSA and 0.02% NaN₃ was added to each tube:

Conc. of Conc. of Gold BSA- Tube # μl mag. beads biotin Biotin Conc. 0 00 0 1 5 1 × 10⁻¹⁴ M 3 × 10⁻⁸ M 8 × 10⁻¹³ M 2 10 2 × 10⁻¹⁴ M 6 ×10^(−8 M) 8 × 10⁻¹³ M 3 15 3 × 10⁻¹⁴ M 9 × 10⁻⁸ M 8 × 10⁻¹³ M 4 20 4 ×10⁻¹⁴ M 12 × 10⁻⁸ M  8 × 10⁻¹³ M

The tubes were incubated for 30 minutes at room temperature and thenwere placed, one at a time starting with tube zero, in a MPC-E/E-1magnetic particle concentrator to separate magnetic beads from solution.After 2 minutes the supernatant solution was carefully removed with thetube still in the magnetic separator. The supernatant was placed in a 1ml microculture tube. Tube 5 was left for 5 minutes in the magneticconcentrator while the other tubes were left for 2 minutes. Thescattered light intensity of the supernates were then measured in theSpectraMetrix photometer with the following settings: Resister PMout=0.1 Meg ohm, Filter on excitation side=orange filter CS 3-67, ×10neutral density attenuator=out.

The following scattered light intensities were measured:

Tube # Intensity 0 1.21 Volts 1 1.04 2 0.644 3 0.49 4 0.362

The supernatants were returned to their respective tubes containing themagnetic beads and allowed to incubate for an additional 2 hours. After2 hours the tubes were then processed as previously described and thefollowing scattered light intensities for the supernates were obtained:

Frac. of Gold Intensity Normalized Particle Tube # (Volts) IntensityBound 0 0.855 1.21 0 1 0.604 0.85 0.3 2 0.382 0.54 0.55 3 0.326 0.460.61 4 0.206 0.29 0.76

The 2 hour extra incubation did not result in greater binding of goldBSA-Biotin to magnetic beads.

A drop of the magnetic beads with attached gold particles was depositedon a microscope glass slide and covered with a cover glass. The slidewas then examined under DLASLPD conditions with a light microscope. Themagnetic beads could easily be seen as strongly scattering objects butthe gold particles on the beads were more difficult to see because ofthe strong scattering by the large magnetic beads. However, if the watermedium was replaced by a bathing medium with a refractive index around1.4 to 1.5, the particles could be seen more clearly. Also if theilluminating light beam was inclined at a higher angle with respect to aline perpendicular to the slide, the gold particles could be seen moreclearly.

Example 32

Detection of Nucleic Acid Hybridization With Nucleic acid-Labeled 40 nmDiameter Gold (Au) Particles

1. Preparation of Chemically Activated Polyethylene Glycol-Amine CoatedAu Particles

Reactive amine groups for conjugation of nucleic acids to the Auparticles was accomplished as follows. 40 nm Au particles were coatedwith bis(Polyoxyethylene bis[3-Amino-2-hydroxypropyl]) Polyethylenecompound using the procedure described in Example 12. This results in 40nm diameter Au particles with a thin coat of polyethylene compound thathas several chemically reactive amine groups for conjugation of thenucleic acids to the particles.

2. Preparation of nucleic acids for conjugation to the 40 nm diameter Auparticles

Homopolymers of polycytidylic acid(Poly (C)) and polyinosinic acid(Poly(I)) were chemically modified as follows. 0.8 mg and 1.3 mg ofPoly(I) and Poly(C) were placed in separate tubes. To each of the tubeswas added 1.0 ml of a 0.1M solution of 1-ethyl-3,3-dimethylaminopropylcarbodiimide (CDI) in imidizole buffer pH 8.5 andincubated for one hour. The nucleic acids were then precipitated byethanol precipitation and resuspended in hybridization buffer (20 mMTris-HCl, 100 mM NaCl, 1 mM EDTA pH 7.6).

3. Conjugation of Activated nucleic Acids to Activated Au Particles

The same protocol was used for both the Poly(I) and Poly(C)preparations. To 50 ul of nucleic acid solution was added 20 ul of 40nm-Au-PEG activated particle solution, and 100 ul of Hepes 0.2 M pH 8.0and incubated for 1 hour at 50° C. Following the reaction, the Poly(C)and Poly(I) 40 nm Au-nucleic acid conjugates were collected bycentrifugation, washed, and resuspended in hybridization buffer.

4. Hybridization Experiments

The hybridization properties of the nucleic acid-40 nm Au particleconjugates (40 nm diameter gold particles with nucleic acids covalentlyattached to the polymer coated surface of the particle) were studied asfollows. A light microscope using DLASLPD methods was used. The glassslide-liquid-cover slip experimental setup as shown in FIG. 9 was used.A drop of the Poly(C)-Au particle conjugate preparation was placed onthe slide and covered with a cover slip. A drop of immersion oil wasplaced on the microscope condenser and the slide then placed on top ofthe condenser. The 10× objective was used. The solution appeared fairlyhomogeneous, and the Poly(C)-Au particle conjugates could be seenfloating across the field. Their brownian motion appeared to be lessthan what we have previously observed for 40 nm Au particles not labeledwith nucleic acids. A few Poly(C)-Au particles appeared to be stuck tothe surface of the glass slide. There were a few aggregates ofPoly(C)-Au particles, mostly of two to four Poly(C)-Au particles. Theseaggregate structures moved as one unit as they floated across the fieldof view. We noticed that the particle density of Poly(C)-Au particlesattached to the surface of the slide was increasing as we observed thisslide for several minutes. The color of the scattered light coming fromthe Poly(C)-Au particles was green. The cover slide was removed, and adrop of the Poly(I)-Au preparation was then placed next to the wet areaof the slide containing the Poly(C)-Au drop. Contact between the twospots was achieved by using a metal probe to drag a line of liquid fromthe Poly(I)-Au drop to the wet part of the slide containing thepoly(C)-Au. A cover slide was then placed on top, and the slide was thenplaced back on the microscope and viewed. We observed the formation ofincreasing numbers of multiple Poly(I)-Au-Poly(C)-Au particle aggregatesover time. After about 20 minutes, we scanned the slide and observedthat there were very few single particles, and most of the particleswere in aggregates of several particles, many of them stuck to the glassslide. The aggregates appeared to have defined shapes, that is, thereseemed to be a particular way these particle aggregates were assembled,some appearing as a spool of randomly wound up string, and othersappeared as branched chain networks. The appearance of these multipleaggregates was very different as compared to the few aggregates weobserved on the control Poly(C)-Au slide. We switched to the 40×objective and in some of the aggregates, some of the particles appearedmore yellow in color than green. We then removed the cover slip of theslide containing the Poly(Au)-Poly(I)-Au reaction and added a drop of10⁻⁵ M ethidium bromide onto the slide and covered with a cover slip. Weobserved a faint orange color coming from the aggregates of particles onthe slides, that is, it appeared like the green and yellow-green colorof the particles was placed on a background of a faint orange color. Noorange color was observed in the areas away from the aggregates. Thisfaint orange color indicated to us that there was double-strandedstructures of nucleic acids near and within the particle aggregates. Weinterpret this as the hybridization of the Poly(C)-Au conjugates toPoly(I)-Au conjugates. This slide was removed and a control slidecontaining a drop of the Poly(I)-Au was observed under the microscope.We made similar observations as compared to the control Poly(C)-Au slidebut that this Poly(I)-Au preparation seemed to have about twice as manysmall aggregates as compared to the Poly(C)-Au control slide. The colorof scattered light appeared green with some aggregates appearingyellow-green.

In this particular format, both of the complementary strands werelabeled with gold particles. As the complementary strands hybridize,more particle aggregates appeared. The binding of nucleic acids can bedetected by detecting the scattered light from gold or similarparticles. It also appears that when two or more Au particles are inclose proximity to each other, the color of the scattered light canchange. This change in color of the scattered light can be used also asa way to detect a binding event. It should be noted that the Auparticles, or any other particle which scatters light sufficiently canbe used in numerous formats to detect the binding of nucleic acids orany other ligand-receptor pair in a separation or non-separation assayformat.

Example 33

Detection of Bound Gold Particles to Large Polystyrene Beads

We placed a drop of a solution of spherical polystyrene particles ofabout 2 microns in diameter coated with biotin on a glass microscopeslide and viewed in the light microscope under DLASLPD conditions. Thepolystyrene particles were easily seen as bright white light pointsources. We then place a drop of a preparation of 60 nm gold particlescoated with streptavidin onto the drop of polystyrene particles andviewed this preparation in the microscope. The bright white polystyreneparticles could be seen and a faint halo of yellow-green color wasobserved surrounding the polystyrene particle. We evaporated thesolution from the slide and then placed a drop of microscope immersionoil on the preparation and then viewed under the microscope. Individualgold particles and large circular ring areas of yellow-green goldparticles were easily visible. The polystyrene particles appeared asalmost a dark or black spot surrounded by a halo or ring of yellow-greencolor. This method can be used to detect bound gold particles or othermetal-like particles to the surface of solid particulate matter andsmall solid-phases such as glass or other beads, and biological cellsand the like.

Example 34

Light Scattering Properties of Gold Particles Coated with PolyethyleneCompound

Gold particles approximately 100 nm in diameter made by the citrateprocedure were used. A portion of this solution was placed in a separatecontainer and the particles were coated with polyethylene glycolcompound(MW=20,000) using the procedure described elsewhere.

For scattered light comparisons of coated and uncoated particles, thesamples were diluted in water until each solution had a faint tinge ofpinkish-red color. The scattered light intensity versus incidentwavelength profiles for the samples were collected using theSpectraMetrix Photometer.

For these measurements, a monochromator was placed between the lightsource and sample Scattered light data was collected at 10 nm incrementsfrom 400 nm to 700 nm by adjusting the monochromator setting. The datawere corrected for wavelength dependent monochromator and photodetectorvariation as a function of wavelength using a calibration graph that wasmade by using 12 nm silica particles. The data was analyzed using thecalibration graph. The data are shown in FIG. 16.

The data show that the coated and uncoated 100 nm gold particles havevery similar scattered light intensity vs. Incident wavelength profiles.Therefore, many different types of macromolecular substances suchantibodies, nucleic acids, receptors, or similar can be coated on thesurface of the particles without significantly altering the scatteredlight properties.

Other embodiments are within the following claims.

What is claimed is:
 1. A method for specific detection of one or more analytes in a sample, comprising the steps of: specifically binding any said one or more analytes in said sample with at least one scattered-light detectable particle of a size between 1 and 500 nm inclusive, illuminating any said particles bound with said analytes with non-evanescent wave light under conditions which produce scattered light from said particle and in which light scattered from one or more said particles can be detected by a human eye with less than 500 times magnification and without electronic amplification, and detecting said light scattered by any said particles under said conditions as a measure of the presence of said one or more analytes, wherein said illuminating and said detecting are on opposite sides of a solid phase surface.
 2. The method of claim 1, wherein said particle has a size which produces a specific colored light when observed by said human eye and illuminated with polychromatic light.
 3. The method of claim 2 wherein the color of said specific colored light provides a measure of the presence or amount of said one or more analytes.
 4. The method of claim 1, wherein said detecting comprises measurement of the intensity of scattered light as a measurement of the presence or amount of said one or more analytes.
 5. The method of claim 1, wherein said detecting comprises measurement of the color of scattered light as a measurement of the presence or amount of said one or more analytes.
 6. The method of claim 1, wherein said particle has a composition which produces a specific colored light when observed by said human eye and illuminated with polychromatic light.
 7. The method of claim 1, wherein said particles are bound to a solid phase bound analyte.
 8. The method of claim 1, wherein said particles are suspended in a liquid phase during said detecting step.
 9. The method of claim 1, wherein said analyte is bound to a solid phase.
 10. The method of claim 9, further comprising a wash step to remove unbound analyte.
 11. The method of claim 1, wherein said analyte is free in liquid solution.
 12. The method of claim 1, wherein said one or more analytes are specifically bound onto a microarray or array chip comprising discrete areas each of which may contain said one or more analytes.
 13. The method of claim 12, wherein said illuminating comprises scanning with a light beam.
 14. The method of claim 13, wherein said scanning is performed by moving an illuminating beam.
 15. The method of claim 13, wherein said scanning is performed by moving the sample.
 16. The method of claim 12, wherein separate spatially addressable sites on said array are separately illuminated.
 17. The method of claim 12, wherein the array is illuminated with a broad light beam.
 18. The method of claim 1, wherein said light is polychromatic light.
 19. The method of claim 1, wherein a monochromatic light illumination source is used to provide said light.
 20. The method of claim 1, wherein said method comprises providing a plurality of different particles each having a different visual appearance when observed by said human eye.
 21. The method of claim 1, wherein said particles are used in a homogeneous assay and wherein two or more particles bound to said one or more analytes are brought sufficiently close together so that the light scattering property of at least one particle is altered by particle—particle perturbations, wherein said alteration is a measure of the presence or amount of said one or more analytes.
 22. The method of claim 1, wherein said particles are used in an assay and wherein two or more particles bound to said one or more analytes are brought sufficiently close together so that the light scattering property of the two or more particles can be resolved from single particles and said light scattering is a measure of the presence or amount of said one more analytes.
 23. The method of claim 1, wherein said particles are used in a homogeneous assay and wherein two or more particles bound to said one or more analytes are brought sufficiently close together so that the light scattering property of the two or more particles can be resolved from single particles and said light scattering is a measure of the presence or amount of said one or more analytes.
 24. The method of claim 1, wherein said particles are used in a homogeneous assay and wherein two or more particles that are held in close proximity to one another are caused to be separated so that the light scattering property of any one particle is altered, wherein said alteration is a measure of the presence or amount of said one or more analytes.
 25. The method of claim 1, wherein said particles are used in a homogeneous assay and wherein two or more particles are linked together by one or more molecular interactions, wherein the molecular interaction holding the particles together is disrupted so that one or more particles is released from the molecular interaction, wherein said released particle or particles is a measure of the presence or amount of said one or more analytes.
 26. The method of claim 1, wherein said particle is a gold or silver particle.
 27. The method of claim 1, wherein said light is directed toward said particle by a prism or other light guide system.
 28. The method of claim 1, wherein said particle is spherical and has a size between 10 and 200 nm inclusive.
 29. The method of claim 1, wherein said particle is spherical and has a size between 20 and 200 nm inclusive.
 30. The method of claim 1, wherein said particle is spherical and has a size between 40 and 120 nm inclusive.
 31. The method of claim 1, wherein said particle is spherical and has a size between 80 and 120 nm inclusive.
 32. The method of claim 1, wherein said particle spherical and has a size between 1 and 10 nm inclusive.
 33. The method of claim 1, wherein said particle spherical and has a size between 11 and 40 nm inclusive.
 34. The method of claim 1, wherein said particle spherical and has a size between 100 and 250 nm inclusive.
 35. The method of claim 1, wherein said particle spherical and has a size greater than 250 nm.
 36. The method of claim 1, wherein said particle is spherical.
 37. The method of claim 1, wherein said particle is ellipsoidal.
 38. The method of claim 1, wherein said particle is asymmetric.
 39. The method of claim 1, wherein said particle comprises a multiple particle aggregate.
 40. The method of claim 1, wherein said illuminating and said detecting are on opposite sides of a solid phase surface.
 41. The method of claim 1 or 40, wherein said illuminating is from below a solid phase surface and said detecting is from above said solid phase surface.
 42. The method of claim 1, wherein said illuminating and said detecting are on the same side of a solid phase surface.
 43. The method of claim 42, wherein said illuminating and said detecting are from above said solid phase surface.
 44. The method of claim 42, wherein said detecting is at an angle to said surface outside the cone of the illuminating light and outside the angle of reflection of the illuminating light.
 45. The method of claim 1, wherein said detecting is at angles outside the angles of incident, transmitted, refracted and reflected light from the illuminating light.
 46. The method of claim 1, wherein said detecting is at right angles to the surface of said sample.
 47. The method of claim 1, wherein said detecting is at angles outside the envelope of forward scattered light.
 48. The method of claim 1, wherein said analyte comprises a protein or peptide.
 49. The method of claim 48, wherein said protein comprises a cell surface constituent.
 50. The method of claim 48, wherein said protein comprises a receptor.
 51. The method of claim 48, wherein said protein comprises an antibody.
 52. The method of claim 1, wherein said one or more analytes comprises one or more analytes on a cell surface, in a cell lysate, or in a chromosome preparation.
 53. The method of claim 1, wherein said analyte comprises an antigen.
 54. The method of claim 1, wherein said analyte comprises a pharmaceutical agent.
 55. The method of claim 1, wherein said one or more analytes comprise combinatorial molecules.
 56. The method of claim 1, wherein said analyte comprises a hormone.
 57. The method of claim 1, wherein said analyte comprises a lipid or carbohydrate.
 58. The method of claim 1, wherein said analyte comprises a nucleic acid molecule.
 59. The method of claim 1, wherein two or more different nucleic acid probe molecules are each bound to said particle, and each said probe will bind to a different target sequence in a nucleic acid analyte.
 60. The method of claim 1, wherein a binding agent is attached to said particles.
 61. The method of claim 60, wherein said binding agent is adsorbed to said particle.
 62. The method of claim 60, wherein said binding agent is covalently attached.
 63. The method of claim 60, wherein said binding agent is attached to a base material.
 64. The method of claim 63, wherein a plurality of different base materials are used.
 65. The method of claim 60, wherein said binding agent is proteinaceous.
 66. The method of claim 65, wherein said binding agent is an antibody.
 67. The method of claim 65, wherein said binding agent is a receptor.
 68. The method of claim 60, wherein said binding agent is a nucleic acid.
 69. The method of claim 60, wherein said binding agent is selected from the group consisting of antigens, lectins, carbohydrates, biotin, avidin, streptavidin, and pharmaceutical agents.
 70. The method of claim 60, wherein said particles are coated with a stabilizing layer.
 71. The method of claim 70, wherein said stabilizing layer is a thin metal coating.
 72. The method of claim 1, wherein said particles comprise coated particles.
 73. The method of claim 72, wherein the coating of said coated particles comprises a stabilizing coating.
 74. The method of claim 73, wherein the coating comprises polymers, proteins, peptides, hormones, antibodies, nucleic acids, or receptors.
 75. The method of claim 73, wherein said stabilizing layer is a thin metal coating.
 76. The method of claim 1, wherein the particles detected are detected following metallographic enlargement.
 77. The method of claim 76, wherein said metallographic enlargement is with gold or silver.
 78. The method of claim 1, further comprising refractive index enhancement of the sensitivity and specificity of detection of said particles.
 79. The method of claim 1, further comprising filtering light through one or more narrow band pass filters.
 80. The method of claim 1, further comprising filtering scattered light through a polarization filter or a bandpass filter or both.
 81. The method of claim 1, wherein said detecting comprises magnification with a microscope 2 to 500 times.
 82. The method of claim 81, wherein said magnification is 10 to 100 times.
 83. The method of claim 81, wherein said detecting comprises use of confocal microscopy.
 84. The method of claim 1, wherein said detecting is by eye.
 85. The method of claim 1, wherein said detecting uses a photodetector.
 86. The method of claim 85, wherein said photodetector comprises a photodiode or photodiode array.
 87. The method of claim 85, wherein said photodetector comprises a photomultiplier tube.
 88. The method of claim 85, wherein said photodetector comprises a camera or video camera.
 89. The method of claim 85, wherein said photodetector comprises a charge-coupled device.
 90. The method of claim 1, wherein said illuminating light is non-polarized.
 91. The method of claim 1, wherein said illuminating light is polarized.
 92. The method of claim 1, wherein said illuminating light is from a filament lamp source.
 93. The method of claim 1, wherein said illuminating light is from a discharge lamp source.
 94. The method of claim 1, wherein said illuminating light is from a laser.
 95. The method of claim 1, wherein said illuminating light is from a light emitting diode.
 96. The method of claim 1, wherein said illuminating light is pulsed.
 97. The method of claim 1, wherein said illuminating light is constant.
 98. The method of claim 1, wherein said illuminating light is coherent.
 99. The method of claim 1, wherein said illuminating light is noncoherent.
 100. The method of claim 1, wherein said analyte is in a sample selected from the group consisting of water, urine, blood, sputum, and tissue.
 101. The method of claim 1, wherein said one or more analytes comprises a plurality of different analytes and said one or more particles comprises a plurality of different particles.
 102. The method of claim 1, wherein said one or more analytes are in a medium comprising particulate matter that scatters light non-specifically to the same or greater intensity as the fluorescence from 10⁻⁹ M fluorescein.
 103. The method of claim 102, wherein said particulate matter in said medium scatters light non-specifically to the same or greater intensity as the fluorescence from 10⁻⁸ M fluorescein.
 104. The method of claim 102, wherein said particulate matter in said medium scatters light non-specifically to the same or greater intensity as the fluorescence from 10⁻⁷ M fluorescein.
 105. The method of claim 1, wherein said particles are mixed composition particles, comprising two or more different materials.
 106. The method of claim 1, further comprising providing spatial filtering to reduce non-specific background light.
 107. The method of claim 1, further comprising providing cutoff filters, narrow bandpass filters, or both to reduce non-specific background light.
 108. The method of claim 1, wherein scattered light from individual particles is detected.
 109. The method of claim 1, wherein scattered light from a plurality of particles is detected.
 110. The method of claim 1, wherein said particle has a size that produces a specific intensity level when illuminated under said conditions.
 111. The method of claim 1, wherein said particle has a size that produces a specific polarization when illuminated under said conditions.
 112. The method of claim 1, wherein said particle has a size that produces a specific angular dependency to said scattered light.
 113. The method of claim 1, wherein said particle has a composition that produces a specific intensity level when illuminated under said conditions.
 114. The method of claim 1, wherein said particle has a composition that produces a specific polarization when illuminated under said conditions.
 115. The method of claim 1, wherein said particle has a composition that produces a specific angular dependency to said scattered light.
 116. The method of claim 1, wherein said one or more analytes is in a microtiter plate.
 117. The method of claim 1, wherein said detecting of said one or more analytes is in a flow-based system.
 118. The method of claim 1, wherein said one or more analytes is in a device selected from the group consisting of a flow cytometry apparatus, a microchannel, and a capillary.
 119. The method of claim 1, wherein said particle is a metal or metal-like particle.
 120. The method of claim 1, wherein the light path of said illuminating or scattered light passes through a refractive index matching material that matches the refractive index of adjacent solid material.
 121. The method of claim 1, wherein said method is performed in a homogeneous format.
 122. The method of claim 1, wherein the light path of said illuminating or scattered light passes through an immersion oil that matches the refractive index of adjacent solid material.
 123. A method for specific detection of one or more analytes in a sample, comprising the steps of: specifically binding any said one or more analytes in said sample with at least one scattered light detectable particles of a size between 1 and 500 nm inclusive, illuminating any said particles bound with said analytes with non-evanescent wave light under conditions which produce scattered light from said particle and in which light scattered from one or more said particles can be detected by a human eye with less than 500 times magnification and without electronic amplification, and detecting said light scattered by any said particles under said conditions as a measure of the presence of said one or more analytes, wherein said detecting is at an angle to the perpendicular to a solid phase surface less than the critical angle, and wherein said illuminating and said detecting are arranged to reduce non-specific background light thereby allowing said specific detection of said one or more analytes bound with said particles. 